Cyclopentane-Based Modulators of STING (Stimulator of Interferon Genes)

ABSTRACT

or a pharmaceutically acceptable salt thereof, processes for the preparation of these compounds, compositions containing these compounds, and the uses of these compounds.

FIELD OF THE INVENTION

This invention relates to novel cyclopentane-based activators of STING(Stimulator of Interferon Genes) useful in the treatment of abnormalcell growth, such as cancer, in mammals. This invention also relates toa method of using such compounds in the treatment of abnormal cellgrowth in mammals, especially humans, and to pharmaceutical compositionsas anticancer agents.

BACKGROUND OF THE INVENTION

The innate immune system is the first line of defense which is initiatedby pattern recognition receptors (PRRs) upon detection of ligands frompathogens as well as damage associated molecular patterns. A growingnumber of these receptors have been identified, which now includessensors of double stranded DNA and unique nucleic acids called cyclicdinucleotides (CDNs). Activation of PRRs leads to up regulation of genesinvolved in the inflammatory response, including type 1 interferons(IFNs), proinflammatory cytokines and chemokines which suppress pathogenreplication and facilitate adaptive immunity.

The adaptor protein STING, also know as TMEM 173, has been identified asa central signalling molecule in the innate immune sensing pathway inresponse to cytosolic nucleic acids. Activation of STING results inup-regulation of IRF3 and NFκB pathways leading to induction of INF-βand other cytokines. STING is critical for responses to cytosolic DNAfrom pathogens or of host origin, and in response to CDNs, sometimereferred to second messengers. G. N. Barber, “Sting: infection,inflammation and cancer,” Nat. Rev. Immun., 2015, 15, pp 760.

CDNs were first identified as bacterial messengers responsible forcontrolling numerous responses in prokaryotic cells. Bacterial CDNs,such as c-di-GMP are symmetrical molecules characterized by two 3′,5′phosphodiester linkages. Direct activation of STING by bacterial CDNshas recently been confirmed through X-ray crystallography. BacterialCDNs have consequently attracted interest as potential vaccineadjuvants.

More recently, the response to cytosolic DNA has been shown to involvegeneration of endogenous CDNs by an enzyme called cyclic guanine adeninesynthase (cGAS), producing a novel mammalian CDN signalling moleculeidentified as cyclic guanine adenine monophosphate (cGAMP), which bindsto and activates STING. Interaction of cGAMP with STING has also beendemonstrated by X-ray crystallography. Unlike bacterial CDNs, cGAMP isan unsymmetrical molecule characterised by its mixed 2′,5′ and 3′,5′phosphodiester linkages. Like bacterial CDNs, cGAMP activates STINGleading to induction of type 1 INFs:

The role of type 1 INFs in response to invading pathogens is wellestablished. Recombinant IFNα was the first approved biologicaltherapeutic and has become an important therapy in viral infections andin cancer. INFs are also know to be potent modulators of the immuneresponse, acting on cells of the immune system.

Given its role in regulating various biological processes, STING is anattractive target for modulation with small molecules. Nevertheless, todate, few effective STING activators have been developed or have enteredthe clinic.

SUMMARY OF THE INVENTION

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type 1 INF and othercytokines, could become an important strategy for the treatment andprevention of human diseases including viral infections and cancer. Thistype of immunomodulatory strategy has the potential to identifycompounds which may be useful not only in infectious diseases but alsocancer, allergic diseases, and as vaccine adjuvants.

Certain compounds of the invention have been shown to bind to STING andto induce type 1 INF and other cytokines and co-stimulatory factors onincubation with human dendritic cells (DCs) and peripheral bloodmononucleocytes (PBMCs). Compounds which induce human INFs may be usefulin the treatment of various disorders, for example the treatment ofallergic diseases and other inflammatory conditions. Certain compoundsof the invention may bind to STING but act as antagonists and thesecould be useful in the treatment of various autoimmune diseases.

It is envisioned that targeting STING with activating or inhibitingagents may be a promising approach for treating diseases and conditionsin which modulation of the typel INF pathway is beneficial, includinginflammatory diseases, allergic and autoimmune diseases, infectiousdiseases, cancer and as vaccine adjuvants.

Each of the embodiments of the non-CDN compounds of the presentinvention described below can be combined with any other embodiment ofthe compounds of the present invention described herein not inconsistentwith the embodiment with which it is combined. Furthermore, each of theembodiments below describing the invention envisions within its scopepharmaceutically acceptable salts of the compounds of the invention.Accordingly, the phrase “or a pharmaceutically acceptable salt thereof”is implicit in the description of all compounds described herein.

The invention includes embodiments wherein there is provided a compoundof formula (I):

or a pharmaceutically acceptable salt thereof, whereineach J is independently oxygen (O) or sulfur (S);R¹ is selected from:

R² is selected from:

W is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;X is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;each Y is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Y substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms;each Z is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Z substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms; andR³ and R⁴ are each independently hydrogen or C₁-C₆ alkyl.

The invention also includes embodiments wherein there is provided acompound of formula (II) (which is a sub-set of formula (I)):

or a pharmaceutically acceptable salt thereof, whereinR¹ is selected from:

R² is selected from:

W is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;X is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;each Y is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Y substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms;each Z is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Z substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms; andR³ and R⁴ are each independently hydrogen or C₁-C₆ alkyl.

Other embodiments of the invention include compounds of formula (I)and/or formula (II) wherein M⁺ is selected from the group consisting ofsodium, potassium, calcium, ammonium, triethylammonium,trimethylammonium and magnesium. Other counter ions are also useful andare included within the scope of the invention. Note that eachcounter-ion M⁺ may be the same, or in some embodiments may be differentfrom one another.

Various combinations of bases R¹ and R² are embodied by the invention.Thus in certain embodiments R¹ is

and R² is

R¹ is

and R² is

wherein R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

R¹ is

and R² is

or R¹ is

and R² is

Other bases (R¹ and R²), and other combinations of bases, are alsoincluded within the present invention.

Additional embodiments of the invention include those where one or bothY is/are halogen. This includes embodiments where one Y is hydrogen andthe other Y is a halogen. In certain of these embodiments the halogen(s)is/are fluorine.

Additional embodiments of the invention include those where one Z ishydrogen and the other Z is OR⁴. In certain of these embodiments one ormore R⁴ is H (hydrogen) and thus at least one Z is OH.

Additional embodiments of the invention include those where W is —SH andX is —SH.

Additional embodiments of the invention include those where W is —OH andX is —OH.

Further embodiments of the invention include a compound selected from:

or a pharmaceutically acceptable salt thereof.

Further embodiments of the invention include a compound selected from:

or a pharmaceutically acceptable salt thereof.

Further embodiments of the invention include a compound selected from:

or a pharmaceutically acceptable salt thereof.

Further embodiments of the invention include a pharmaceuticalcomposition comprising a compound or salt as described herein, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. Optionally, such compositions may comprise acompound or salt as described herein which is a component of anantibody-drug conjugate; and/or may comprise a compound as describedherein which is a component of a particle-based delivery system.

Also embodied in the invention is a method of treating abnormal cellgrowth in a mammal, the method comprising administering to the mammal atherapeutically effective amount of a compound or salt as describedherein. This method may (or may not) employ a compound or salt asdescribed herein as a component of an antibody-drug conjugate, or as acomponent of a particle-based delivery system. In such embodiments theabnormal cell growth may be cancer. If the abnormal cell growth iscancer, the cancer to be treated may be lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, lymphocytic lymphomas,cancer of the bladder, cancer of the kidney or ureter, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, or pituitary adenoma.

Also embodied in the invention is the use of a compound or salt asdescribed herein for the preparation of a medicament useful in thetreatment of abnormal cell growth in a mammal. In such embodiments theabnormal cell growth may be cancer. If the abnormal cell growth iscancer, the cancer to be treated may be lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, lymphocytic lymphomas,cancer of the bladder, cancer of the kidney or ureter, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, or pituitary adenoma.

Further still, embodiments of the invention include those where there isprovided a method of upregulating the activity of STING in a mammal,comprising the step of administering to said mammal an effective amountof a compound or salt as described herein; and/or a method of increasinginterferon-beta levels in a mammal, comprising the step of administeringto said mammal an effective amount of a compound or salt as describedherein.

Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the meanings discussed below. Variables defined in thissection, such as R, X, n and the like, are for reference within thissection only, and are not meant to have the same meaning as may be usedoutside of this definitions section. Further, many of the groups definedherein can be optionally substituted. The listing in this definitionssection of typical substituents is exemplary and is not intended tolimit the substituents defined elsewhere within this specification andclaims.

“Alkenyl” refers to an alkyl group, as defined herein, consisting of atleast two carbon atoms and at least one carbon-carbon double bond.Representative examples include, but are not limited to, ethenyl,1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like. “Alkenylene”refers to a di-valent form of alkenyl.

“Alkoxy” refers to —O-alkyl where alkyl is preferably C₁-C₈, C₁-C₇,C₁-C₆, C₁-C₅, C₁-C₄, C₁-C₃, C₁-C₂ or C₁ alkyl.

“Alkyl” refers to a saturated aliphatic hydrocarbon radical includingstraight chain and branched chain groups of 1 to 20 carbon atoms(“(C₁-C₂₀)alkyl”), preferably 1 to 12 carbon atoms (“(C₁-C₁₂)alkyl”),more preferably 1 to 8 carbon atoms (“(C₁-C₈)alkyl”), or 1 to 6 carbonatoms (“(C₁-C₆)alkyl”), or 1 to 4 carbon atoms (“(C₁-C₄)alkyl”).Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl,n-butyl, iso-butyl, tert-butyl, pentyl, neopentyl, and the like. Alkylmay be substituted or unsubstituted. Typical substituent groups includecycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl,O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, C-carboxy, O-carboxy, nitro, silyl, amino and —NR^(x)R^(y),where R^(x) and R^(y) are for example hydrogen, alkyl, cycloalkyl, aryl,carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and, combined, afive- or six-member heteroalicyclic ring. “Haloalkyl” for instance(C₁-C₈)haloalkyl, refers to an alkyl having one or more, halogensubstituents. “Alkylene” refers to a di-valent form of alkyl.

“Alkynyl” refers to an alkyl group, as defined herein, consisting of atleast two carbon atoms and at least one carbon-carbon triple bond.Representative examples include, but are not limited to, ethynyl,1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like. “Alkynylene”refers to a di-valent form of alkynyl.

“Amino” refers to an —NR^(x)R^(y) group, wherein R^(x) and R^(y) areboth hydrogen.

“Cyano” refers to a —C≡N group. Cyano may be expressed as CN.

“(C₃-C₅)cycloalkyl” refers to a 3 to 5 member all-carbon monocyclicring. Examples, without limitation, of cycloalkyl groups arecyclopropane, cyclobutane, cyclopentane, and cyclopentene. Typicalsubstituent groups include alkyl, alkoxy, cyano, halo,carbonylC-carboxy, O-carboxy, O-carbamyl, N-carbamyl, amino and—NR^(x)R^(y), with R^(x) and R^(y) as defined above. Illustrativeexamples of cycloalkyl are derived from, but not limited to, thefollowing:

“Halogen” or the prefix “halo” refers to fluoro, chloro, bromo and iodo.Preferably halogen refers to fluoro or chloro.

“Heteroatom” refers to an atom selected from the group consisting of O,N, Si, S and/or P, and wherein the nitrogen and sulfur atoms mayoptionally be oxidized.

“Heterocyclyl” refers to a monocyclic or fused ring system having 3 to12 ring atoms containing one, two, three or four ring heteroatomsselected from N, O, and S(O)_(n) (where n is 0, 1 or 2), and 1-9 carbonatoms The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system. Preferredheterocycles include (C₂-C₆)heterocycles in accordance with thedefinition above. Examples of suitable saturated heteroalicyclic groupsinclude, but are not limited to:

The heterocyclyl group is optionally substituted with one or twosubstituents independently selected from halo, lower alkyl.

“Hydroxy” or “hydroxyl” refers to an —OH group.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium.

“In vivo” refers to procedures performed within a living organism suchas, without limitation, a mouse, rat, rabbit and/or human.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “heterocycle group optionallysubstituted with an alkyl group” means that the alkyl may but need notbe present, and the description includes situations where theheterocycle group is substituted with an alkyl group and situationswhere the heterocycle group is not substituted with the alkyl group.

“Organism” refers to any living entity comprised of at least one cell. Aliving organism can be as simple as, for example, a single eukarioticcell or as complex as a mammal, including a human being.

A “pharmaceutically acceptable excipient” refers to an inert substanceadded to a pharmaceutical composition to further facilitateadministration of a compound. Examples, without limitation, ofexcipients include calcium carbonate, calcium phosphate, various sugarsand types of starch, cellulose derivatives, gelatin, vegetable oils andpolyethylene glycols.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which retain the biological effectiveness and properties ofthe parent compound. Such salts include:

(i) acid addition salts, which can be obtained by reaction of the freebase of the parent compound with inorganic acids such as hydrochloricacid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, andperchloric acid and the like, or with organic acids such as acetic acid,oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaricacid, citric acid, succinic acid or malonic acid and the like; or

(ii) salts formed when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, trialkylamonium and the like.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or physiologically/pharmaceuticallyacceptable salts, solvates, hydrates or prodrugs thereof, with otherchemical components, such as physiologically/pharmaceutically acceptablecarriers and excipients. The purpose of a pharmaceutical composition isto facilitate administration of a compound to an organism.

As used herein, a “physiologically/pharmaceutically acceptable carrier”refers to a carrier or diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound.

“Therapeutically effective amount” refers to that amount of the compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disorder being treated. In reference to the treatment ofcancer, a therapeutically effective amount refers to that amount whichhas at least one of the following effects:

-   -   (1) reducing the size of the tumor;    -   (2) inhibiting (that is, slowing to some extent, preferably        stopping) tumor metastasis;    -   (3) inhibiting to some extent (that is, slowing to some extent,        preferably stopping) tumor growth, and    -   (4) relieving to some extent (or, preferably, eliminating) one        or more symptoms associated with the cancer.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabrogating a cellular disorder and/or its attendant symptoms. Withregard particularly to cancer, these terms simply mean that the lifeexpectancy of an individual affected with a cancer will be increased orthat one or more of the symptoms of the disease will be reduced.

DETAILED DESCRIPTION

General schemes for synthesizing the compounds of the invention can befound in the Examples section herein.

Unless indicated otherwise, all references herein to the inventivecompounds include references to salts, solvates, hydrates and complexesthereof, and to solvates, hydrates and complexes of salts thereof,including polymorphs, stereoisomers, and isotopically labeled versionsthereof.

Pharmaceutically acceptable salts include acid addition and base salts(including disalts).

Suitable acid addition salts are formed from acids which form non-toxicsalts. Examples include the acetate, aspartate, benzoate, besylate,bicarbonate/carbonate, bisulphate/sulfate, borate, camsylate, citrate,edisylate, esylate, formate, fumarate, gluceptate, gluconate,glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate,succinate, tartrate, tosylate and trifluoroacetate salts.

Suitable base salts are formed from bases which form non-toxic salts.Examples include the aluminum, arginine, benzathine, calcium, choline,diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine,potassium, sodium, tromethamine and zinc salts. For a review on suitablesalts, see “Handbook of Pharmaceutical Salts: Properties, Selection, andUse” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002), thedisclosure of which is incorporated herein by reference in its entirety.

A pharmaceutically acceptable salt of the inventive compounds can bereadily prepared by mixing together solutions of the compound and thedesired acid or base, as appropriate. The salt may precipitate fromsolution and be collected by filtration or may be recovered byevaporation of the solvent. The degree of ionization in the salt mayvary from completely ionized to almost non-ionized.

The compounds of the invention may exist in both unsolvated and solvatedforms. The term ‘solvate’ is used herein to describe a molecular complexcomprising the compound of the invention and one or morepharmaceutically acceptable solvent molecules, for example, ethanol. Theterm ‘hydrate’ is employed when the solvent is water. Pharmaceuticallyacceptable solvates in accordance with the invention include hydratesand solvates wherein the solvent of crystallization may be isotopicallysubstituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

Also included within the scope of the invention are complexes such asclathrates, drug-host inclusion complexes wherein, in contrast to theaforementioned solvates, the drug and host are present in stoichiometricor non-stoichiometric amounts. Also included are complexes of the drugcontaining two or more organic and/or inorganic components which may bein stoichiometric or non-stoichiometric amounts. The resulting complexesmay be ionized, partially ionized, or non-ionized. For a review of suchcomplexes, see J Pharm Sci, 64 (8), 1269-1288 by Haleblian (August1975), the disclosure of which is incorporated herein by reference inits entirety.

Also within the scope of the invention are polymorphs, prodrugs, andisomers (including optical, geometric and tautomeric isomers) of theinventive compounds

Derivatives of compounds of the invention which may have little or nopharmacological activity themselves but can, when administered to apatient, be converted into the inventive compounds, for example, byhydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’.Further information on the use of prodrugs may be found in ‘Pro-drugs asNovel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and WStella) and ‘Bioreversible Carriers in Drug Design’, Pergamon Press,1987 (ed. E B Roche, American Pharmaceutical Association), thedisclosures of which are incorporated herein by reference in theirentireties.

Prodrugs in accordance with the invention can, for example, be producedby replacing appropriate functionalities present in the inventivecompounds with certain moieties known to those skilled in the art as‘pro-moieties’ as described, for example, in “Design of Prodrugs” by HBundgaard (Elsevier, 1985), the disclosure of which is incorporatedherein by reference in its entirety.

Some examples of prodrugs in accordance with the invention include:

(i) where the compound contains a carboxylic acid functionality —(COOH),an ester thereof, for example, replacement of the hydrogen with(C₁-C₈)alkyl;

(ii) where the compound contains an alcohol functionality (—OH), anether thereof, for example, replacement of the hydrogen with(C₁-C₆)alkanoyloxymethyl; and

(iii) where the compound contains a primary or secondary aminofunctionality (—NH₂ or —NHR where R≠H), an amide thereof, for example,replacement of one or both hydrogens with (C₁-C₁₀)alkanoyl.

Further examples of replacement groups in accordance with the foregoingexamples and examples of other prodrug types may be found in theaforementioned references.

Finally, certain inventive compounds may themselves act as prodrugs ofother of the inventive compounds.

Compounds of the invention containing one or more asymmetric carbonand/or phosphorous atoms can exist as two or more stereoisomers. Wherethe compounds according to this invention have at least one chiralcenter, they may accordingly exist as enantiomers. Where the compoundspossess two or more chiral centers, they may additionally exist asdiastereomers. Similarly, where a compound of the invention contains acyclopropyl group or other cyclic group where chirality exists, andalkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers arepossible. Where the compound contains, for example, a keto or oximegroup or an aromatic moiety, tautomeric isomerism (‘tautomerism’) canoccur. A single compound may exhibit more than one type of isomerism.

Included within the scope of the invention are all stereoisomers,geometric isomers and tautomeric forms of the inventive compounds,including compounds exhibiting more than one type of isomerism, andmixtures of one or more thereof. Also included are acid addition or basesalts wherein the counterion is optically active, for example, D-lactateor L-lysine, or racemic, for example, DL-tartrate or DL-arginine.

Cis/trans isomers may be separated by conventional techniques well knownto those skilled in the art, for example, chromatography and fractionalcrystallization.

Conventional techniques for the preparation/isolation of individualenantiomers include chiral synthesis from a suitable optically pureprecursor or resolution of the racemate (or the racemate of a salt orderivative) using, for example, chiral high pressure liquidchromatography (HPLC) or supercritical fluid chromatography (SFC).

Alternatively, the racemate (or a racemic precursor) may be reacted witha suitable optically active compound, for example, an alcohol, or, inthe case where the compound contains an acidic or basic moiety, an acidor base such as tartaric acid or 1-phenylethylamine. The resultingdiastereomeric mixture may be separated by chromatography and/orfractional crystallization and one or both of the diastereoisomersconverted to the corresponding pure enantiomer(s) by means well known toone skilled in the art.

Stereoisomeric conglomerates may be separated by conventional techniquesknown to those skilled in the art; see, for example, “Stereochemistry ofOrganic Compounds” by E L Eliel (Wiley, New York, 1994), the disclosureof which is incorporated herein by reference in its entirety.

The invention also includes isotopically-labeled compounds of theinvention, wherein one or more atoms is replaced by an atom having thesame atomic number, but an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopessuitable for inclusion in the compounds of the invention includeisotopes of hydrogen, such as ²H and ³H, carbon, such as ¹¹C, ¹³C and¹⁴C, chlorine, such as ³⁶Cl, fluorine, such as ¹⁸F, iodine, such as ¹²³Iand ¹²⁵I, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and¹⁸O, phosphorus, such as ³²P, and sulfur, such as ³⁵S. Certainisotopically-labeled compounds of the invention, for example, thoseincorporating a radioactive isotope, are useful in drug and/or substratetissue distribution studies. The radioactive isotopes tritium, ³H, andcarbon-14, ¹⁴C, are particularly useful for this purpose in view oftheir ease of incorporation and ready means of detection. Substitutionwith heavier isotopes such as deuterium, ²H, may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence may be preferred in some circumstances. Substitution with positronemitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful inPositron Emission Topography (PET) studies for examining substratereceptor occupancy.

Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed.

Pharmaceutically acceptable solvates in accordance with the inventioninclude those wherein the solvent of crystallization may be isotopicallysubstituted, e.g. D₂O, d₆-acetone, d₆-DMSO.

Compounds of the invention intended for pharmaceutical use may beadministered as crystalline or amorphous products, or mixtures thereof.They may be obtained, for example, as solid plugs, powders, or films bymethods such as precipitation, crystallization, freeze drying, spraydrying, or evaporative drying. Microwave or radio frequency drying maybe used for this purpose.

The compounds can be administered alone or in combination with one ormore other compounds of the invention. Generally, they will beadministered as a formulation in association with one or morepharmaceutically acceptable excipients. The term “excipient” is usedherein to describe any ingredient other than the compound(s) of theinvention. The choice of excipient will to a large extent depend onfactors such as the particular mode of administration, the effect of theexcipient on solubility and stability, and the nature of the dosageform.

The compositions described herein can be administered to a host, eitheralone or in combination with a pharmaceutically acceptable excipient, inan amount sufficient to induce, modify, or stimulate an appropriateimmune response. The immune response can comprise, without limitation,specific immune response, non-specific immune response, both specificand non-specific response, innate response, primary immune response,adaptive immunity, secondary immune response, memory immune response,immune cell activation, immune cell proliferation, immune celldifferentiation, and cytokine expression. In certain embodiments, thecompositions are administered in conjunction with one or more additionalcompositions including vaccines intended to stimulate an immune responseto one or more predetermined antigens; adjuvants; CTLA-4 and PD-1pathway antagonists, lipids, liposomes, chemotherapeutic agents,immunomodulatory cell lines, etc.

In some aspects of the invention, the methods described herein furtherinclude a step of treating a subject with an additional form of therapy.In some aspects, the additional form of therapy is an additionalanti-cancer therapy including, but not limited to, chemotherapy,radiation, surgery, hormone therapy, and/or additional immunotherapy.

The disclosed STING modulatory compounds may be administered as aninitial treatment, or for treatment of cancers that are unresponsive toconventional therapies. In addition, the disclosed STING modulatorycompounds may be used in combination with other therapies (e.g.,surgical excision, radiation, additional anti-cancer drugs etc.) tothereby elicit additive or potentiated therapeutic effects and/or reducecytotoxicity of some anti-cancer agents. The STING modulatory compoundsof the invention may be co-administered or co-formulated with additionalagents, or formulated for consecutive administration with additionalagents in any order.

The STING modulatory compounds the invention may be used in combinationwith other therapeutic agents including, but not limited to, therapeuticantibodies, ADCs, immunomodulating agents, cytotoxic agents, andcytostatic agents. A cytotoxic effect refers to the depletion,elimination and/or the killing of a target cells (i.e., tumor cells). Acytotoxic agent refers to an agent that has a cytotoxic and/orcytostatic effect on a cell. A cytostatic effect refers to theinhibition of cell proliferation. A cytostatic agent refers to an agentthat has a cytostatic effect on a cell, thereby inhibiting the growthand/or expansion of a specific subset of cells (i.e., tumor cells). Animmunomodulating agent refers to an agent that stimulates the immuneresponse though the production of cytokines and/or antibodies and/ormodulating T cell function thereby inhibiting or reducing the growth ofa subset of cells (i.e., tumor cells) either directly or indirectly byallowing another agent to be more efficacious.

For combination therapies, the STING modulatory compounds areadministered within any time frame suitable for performance of theintended therapy. Thus, the single agents may be administeredsubstantially simultaneously (i.e., as a single formulation or withinminutes or hours) or consecutively in any order. For example, singleagent treatments may be administered within about 1 year of each other,such as within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or 1week(s), or within about 5, 4, 3, 2 or 1 day(s).

The disclosed combination therapies may elicit a synergistic therapeuticeffect, i.e., an effect greater than the sum of their individual effectsor therapeutic outcomes. For example, a synergistic therapeutic effectmay be an effect of at least about two-fold greater than the therapeuticeffect elicited by a single agent, or the sum of the therapeutic effectselicited by the single agents of a given combination, or at least aboutfive-fold greater, or at least about ten-fold greater, or at least abouttwenty-fold greater, or at least about fifty-fold greater, or at leastabout one hundred-fold greater. A synergistic therapeutic effect mayalso be observed as an increase in therapeutic effect of at least 10%compared to the therapeutic effect elicited by a single agent, or thesum of the therapeutic effects elicited by the single agents of a givencombination, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or more. A synergistic effect is also aneffect that permits reduced dosing of therapeutic agents when they areused in combination.

The compositions may be administered before, after, and/or together withan additional therapeutic or prophylactic composition or modality. Theseinclude, without limitation, B7 costimulatory molecule, interleukin-2,interferon-7, GM-CSF, CTLA-4 antagonists, OX-40/OX-40 ligand, CD40/CD40ligand, sargramostim, levamisol, vaccinia virus, Bacille Calmette-Guerin(BCG), liposomes, alum, Freund's complete or incomplete adjuvant,detoxified endotoxins, mineral oils, surface active substances such aslipolecithin, pluronic polyols, polyanions, peptides, and oil orhydrocarbon emulsions. Carriers for inducing a T cell immune responsewhich preferentially stimulate a cytolytic T cell response versus anantibody response are preferred, although those that stimulate bothtypes of response can be used as well. In cases where the agent is apolypeptide, the polypeptide itself or a polynucleotide encoding thepolypeptide can be administered. The carrier can be a cell, such as anantigen presenting cell (APC) or a dendritic cell. Antigen presentingcells include such cell types as macrophages, dendritic cells andBcells. Other professional antigen-presenting cells include monocytes,marginal zone Kupffer cells, microglia, Langerhans' cells,interdigitating dendritic cells, follicular dendritic cells, and Tcells. Facultative antigen-presenting cells can also be used. Examplesof facultative antigenpresenting cells include astrocytes, follicularcells, endothelium and fibroblasts. The carrier can be a bacterial cellthat is transformed to express the polypeptide or to deliver apolynucleotide which is subsequently expressed in cells of thevaccinated individual. Adjuvants, such as aluminium hydroxide oraluminum phosphate, can be added to increase the ability of the vaccineto trigger, enhance, or prolong an immune response. Additionalmaterials, such as cytokines, chemokines, and bacterial nucleic acidsequences, like CpG, a toll-like receptor (TLR) 9 agonist as well asadditional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9,including lipoprotein, LPS, monophosphoryl lipid A, lipoteichoic acid,imiquimod, resiquimod, and in addition retinoic acid-inducible gene I(RIG-I) agonists such as poly I:C, used separately or in combinationwith the described compositions are also potential adjuvants. Otherrepresentative examples of adjuvants include the synthetic adjuvantQS-21 comprising a homogeneous saponin purified from the bark ofQuillaja saponaria and Colynebacterium parvum (McCune et al., Cancer,1979; 43:1619). It will be understood that the adjuvant is subject tooptimization. In other words, the skilled artisan can engage in routineexperimentation to determine the best adjuvant to use.

Pharmaceutical compositions suitable for the delivery of compounds ofthe invention and methods for their preparation will be readily apparentto those skilled in the art. Such compositions and methods for theirpreparation can be found, for example, in ‘Remington's PharmaceuticalSciences’, 19th Edition (Mack Publishing Company, 1995), the disclosureof which is incorporated herein by reference in its entirety.

The compounds of the invention may be administered directly into theblood stream, into muscle, or into an internal organ. Suitable means forparenteral administration include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, subcutaneous andintratumoral. Suitable devices for parenteral administration includeneedle (including micro needle) injectors, needle-free injectors andinfusion techniques.

Parenteral formulations are typically aqueous solutions which maycontain excipients such as salts, carbohydrates and buffering agents(preferably to a pH of from 3 to 9), but, for some applications, theymay be more suitably formulated as a sterile non-aqueous solution or asa dried form to be used in conjunction with a suitable vehicle such assterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, forexample, by lyophilization, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.

The solubility of compounds of the invention used in the preparation ofparenteral solutions may be increased by the use of appropriateformulation techniques, such as the incorporation ofsolubility-enhancing agents. Formulations for parenteral administrationmay be formulated to be immediate and/or modified release. Modifiedrelease formulations include delayed-, sustained-, pulsed-, controlled-,targeted and programmed release. Thus compounds of the invention may beformulated as a solid, semi-solid, or thixotropic liquid foradministration as an implanted depot providing modified release of theactive compound. Examples of such formulations include drug-coatedstents and PLGA microspheres.

Nanoparticles also represent drug delivery systems suitable for mostadministration routes. Over the years, a variety of natural andsynthetic polymers have been explored for the preparation ofnanoparticles, of which Poly(lactic acid) (PLA), Poly(glycolic acid)(PGA), and their copolymers (PLGA) have been extensively investigatedbecause of their biocompatibility and biodegradability. Nanoparticlesand other nanocarriers act as potential carries for several classes ofdrugs such as anticancer agents, antihypertensive agents,immunomodulators, and hormones; and macromolecules such as nucleicacids, proteins, peptides, and antibodies. See, e.g., Crit. Rev. Ther.Drug Carrier Syst. 21:387-422, 2004; Nanomedicine: Nanotechnology,Biology and Medicine 1:22-30, 2005.

The compositions of the present invention may comprise, or beadministered together with, one or more additional pharmaceuticallyactive components such as adjuvants, lipids, interbilayer crosslinkedmultilamellar vesicles, biodegradable poly(D, L-lactic-co-glycolic acid)[PLGA]-based or poly anhydride-based nanoparticles or microparticles,and nanoporous particle-supported lipid bilayers such as liposomes,CTLA-4 and PD-1 pathway Antagonists, PD-1 pathway blocking agents,inactivated bacteria which induce innate immunity (e.g., inactivated orattenuated Listeria monocytogenes), compositions which mediate innateimmune activation via Toll-like Receptors (TLRs), (NOD)-like receptors(NLRs), Retinoic acid inducible gene-based (RIG)-1-like receptors(RLRs), C-type lectin receptors (CLRs), pathogenassociated molecularpatterns (“PAMPs”), chemotherapeutic agents, and the like.

The compounds and compositions of the present invention may beadministered as a component of an antibody-drug conjugate or othertargeted delivery modality.

Topical Administration

Compounds of the invention may be combined with soluble macromolecularentities, such as cyclodextrin and suitable derivatives thereof orpolyethylene glycol-containing polymers, in order to improve theirsolubility, dissolution rate, taste-masking, bioavailability and/orstability for use in any of the aforementioned modes of administration.

Drug-cyclodextrin complexes, for example, are found to be generallyuseful for most dosage forms and administration routes. Both inclusionand non-inclusion complexes may be used. As an alternative to directcomplexation with the drug, the cyclodextrin may be used as an auxiliaryadditive, i.e. as a carrier, diluent, or solubilizer. Most commonly usedfor these purposes are alpha-, beta- and gamma-cyclodextrins, examplesof which may be found in PCT Publication Nos. WO 91/11172, WO 94/02518and WO 98/55148, the disclosures of which are incorporated herein byreference in their entireties.

Dosage: The amount of the active compound administered will be dependenton the subject being treated, the severity of the disorder or condition,the rate of administration, the disposition of the compound and thediscretion of the prescribing physician. One possible dosage is in therange of about 0.001 to about 100 mg per kg body weight, administereddaily, every other day, every third day, every fourth day, every fifthday, every sixth day, weekly, every other week, monthly, or on otherdosing schedules. In some instances, dosage levels below the lower limitof the aforesaid range may be more than adequate, while in other casesstill larger doses may be used without causing any harmful side effect,with such larger doses typically divided into several smaller doses foradministration throughout the day.

Kit-of-Parts: Inasmuch as it may desirable to administer a combinationof active compounds, for example, for the purpose of treating aparticular disease or condition, it is within the scope of the presentinvention that two or more pharmaceutical compositions, at least one ofwhich contains a compound in accordance with the invention, mayconveniently be combined in the form of a kit suitable forcoadministration of the compositions. Thus the kit of the inventionincludes two or more separate pharmaceutical compositions, at least oneof which contains a compound of the invention, and means for separatelyretaining said compositions, such as a container, divided bottle, ordivided foil packet. An example of such a kit is the familiar blisterpack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administeringdifferent dosage forms, for example, oral and parenteral, foradministering the separate compositions at different dosage intervals,or for titrating the separate compositions against one another. Toassist compliance, the kit typically includes directions foradministration and may be provided with a memory aid.

EXAMPLES General Methods Synthetic Experimental Procedures

Experiments were generally carried out under inert atmosphere (nitrogenor argon), particularly in cases where oxygen- or moisture-sensitivereagents or intermediates were employed. Commercial solvents andreagents were generally used without further purification and dried overmolecular sieves (generally Sure-Seal™ products from the AldrichChemical Company, Milwaukee, Wis.). Mass spectrometry data is reportedfrom either liquid chromatography-mass spectrometry (LC-MS), atmosphericpressure chemical ionization (APCI), electrospray ionization (ESI) orliquid chromatography-Time of Flight (LC-TOF) methods. Chemical shiftsfor nuclear magnetic resonance (NMR) data are expressed in parts permillion (ppm) referenced to residual peaks from the deuterated solventsemployed.

For syntheses referencing procedures in other Examples or Methods,reaction Protocol (length of reaction and temperature) may vary. Ingeneral, reactions were followed by thin layer chromatography, LC-MS orHPLC, and subjected to work-up when appropriate. Purifications may varybetween experiments: in general, solvents and the solvent ratios usedfor eluents/gradients were chosen to provide appropriate retentiontimes. Unless otherwise specified, reverse phase HPLC fractions wereconcentrated via lyophilization/freeze-drying. Intermediate and finalcompounds were stored at (0° C.) or room temperature in closed vials orflasks under nitrogen. Compound names were generated with Chemdraw orACD Labs software.

Abbreviations for solvents and/or reagents are based on AmericanChemical Society guidelines and are highlighted below:

Ac=Acetyl; Boc=N-tert-butoxycarbonyl; BTT=Benzylthiotetrazole;CDI=N,N′-Carbonyldiimidazole; DCA=Dichloroacetic acid;DCC=1,3-Dicyclohexylcarbodiimide; DCE=Dichloroethane;DCM=Dichloromethane;DDTT=(E)-N,N-Dimethyl-N′-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide;DEA=N,N-Diethylamine; DIBAL-H=Diisobutylaluminium hydride;DIPEA=N,N-Diisopropylethylamine; DMA=Dimethylacetamide;DMAP=4-Dimethylaminopyridine; DME=Dimethoxyethane;DMF=N,N-Dimethylformamide;DMOCP=2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide;DMSO=Dimethyl sulfoxide; DMT=Dimethoxytrityl; DMTCl=Dimethoxytritylchloride; DPPA=Diphenylphosphoryl azide;EDCI=1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EtOAc=Ethyl acetate;ETT=Ethylthiotetrazole; Fmoc=Fluorenylmethyloxycarbonyl; h=hour;HATU=o-(7-azabenzotriazol-1-yl)-N,N,N′,N′-te-tramethyluroniumhexafluorophosphate;HBTU=N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate; HOAc=Acetic acid;HOAt=1-Hydroxy-7-azabenzotriazole; HOBt=1-Hydroxybenzotriazole hydrate;LDA=Lithium diisopropylamide; Me=Methyl; MTBE=Methyl tert-butyl ether;n-BuLi=n-Butyllithium; NBS=N-Bromosuccinimide; NMM=N-methyl morpholine;NMO=N-methyl morpholine N-oxide; Ph=Phenyl; PivCl=Pivaloyl chloride;PPTS=Pyridinium p-Toluenesulfonate; p-TsOH=p-Toluenesulfonic acid;rt=room temperature; TEAB=Tetraethylammonium Bromide;TBAI=Tetrabutylammonium Iodide; TBS=tert-Butyldimethylsilyl;TBSCl=tert-Butyldimethylsilyl Chloride; TEA=Triethylamine;Tf=Trifluoromethanesulfonate; TFA=Trifluoroacetic acid;THF=Tetrahydrofuran; andTPTU=O-(2-Oxo-1(2H)pyridyl)-N,N,N,′N′-tetramethyluroniumtetrafluoroborate.

General Scheme

In general, the synthesis of the cyclopentane-based STING activators canbe analogized to the routes used to make the appropriate macrocyclesfound in the synthesis of cyclic dinuclueotides (Gaffney B. L., et. al.;Organic Letters 2010 12(14) 3269-3271).

As exemplified in Scheme 1, the chiral allylic acetate 1a can bepurchased or synthesized (Deardorff D., et. al.; Tetrahedron Letters1986 27(11) 1255-1256). An allylic alkylation can be performed with anitrogen contain heterocycle or nucleobase to form compounds such as 1b(Trost, B., et. al.; Angew. Chem. Int. Ed. 1996, 35 1569-1572). Thesereactions are typically run using a palladium catalyst, a phosphineligand and basic condition. Other metal catalyst and ligand combinationcan also be employed to achieve the same transformation. Typically,protection of the allylic alcohol, 1b, with a dimethoxytrityl (DMT)group is achieved using dimethoxytrityl chloride (DMTCl) and a base togive compounds such as 1c. The double bond of 1c is typicallydihydroxylated with catalytic osmium tetroxide and N-methylpiperidineN-oxide to give compounds such as 1d. Other dihydroxylating reagentssuch as permanganate or ruthenium tetroxide may be used to do the sametransformation. Typically, mono-protection of the compounds such as 1dcan be achieved using tetrabutyldimethylsilyl choride (TBSCl) withtetrazole or tetrabutyldimethylsilyl trifluoromethanesulfonate (TBSOTf)and base to give compounds such as 1e. Phosphoramidites such as compound1f are typically made from compounds such as 1e when treated with3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile and base.H-phosphonates and thio-H-phosphonates such as compound 1g are generallymade from the appropriately protected nucleoside and a mixed anhydrideof phosphonic acid followed by sulfurization if necessary. Theprotecting group is removed to reveal the primary alcohol 1g. Couplingof compounds such as 1g and 1f generally occur after phosphoramiditessuch as 1f are treated with acidic activators. The resulting coupledmaterial is then sulfurized with sulfurizing agents such as DDTT,3H-benzodithiol-3-one or similar reagent to produce thiophosphonatessuch as 1h. Otherwise the coupled material can be oxidized with reagentssuch as t-butyl peroxide or similar oxidants to produce phosphates suchas 1h. Compounds such as 1h can be treated with mild acid such asdichloroacetic acid to reveal compounds such as 1i. H-phosphonatecompounds such as 1i can be activated with reagents such as DMOCP toaffect macrocyclization which then can be sulfurized by reagents such as3H-benzodithiol-3-one to generate cyclic thiophosphonates such as 1j oroxidized with reagents such as t-butylperoxide to generate cyclicphosphates such as 1j. After the appropriate deprotection conditions,cyclic dithiophosphonates, diphosphates or mixedthiophosphonate-phosphate compounds such as 1k can be generated.Compounds at every step may be purified by standard techniques such ascolumn chromatography, crystallization, reverse phase HPLC or SFC. Ifnecessary, separation of the diastereomers of 1k may be carried outunder standard methods known in the art such as chiral SFC or HPLC toafford single diastereomers. Note that “A” denotes carbon (also bound tohydrogen or a substituent) or nitrogen.

Example 1 Synthesis of(4S,6R,7S,11aR,13R,14R,14aR,15R)-6,13-bis(6-amino-9H-purin-9-yl)-14-fluoro-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-15-ol2,9-dioxide

Step 1: Synthesis ofN-(9-((1R,4S)-4-hydroxycyclopent-2-en-1-yl)-9H-purin-6-yl)benzamide(A-3)

To an oven dried round bottom flask (flask A), equipped with a magneticstirbar and purged with N₂, was added A-1 (8330 mg, 34.8 mmol) and DMF(50 mL). To the solution was added NaH (60 wt % dispersion in mineraloil, 1530 mg, 38.3 mmol) under N₂. To a second round bottom flask (flaskB), equipped with a magnetic stirbar and purged with N₂, was added A-2(4950 mg, 34.8 mmol), Pd(PPh₃)₄ (2490 mg, 2.16 mmol), PPh₃ (913 mg, 3.48mmol), and THF (50 mL). After 30 min, the solution in flask A wastransferred to flask B. Flask B was then placed in an oil bath andheated at 50° C. under N₂ for 12 hours. The reaction mixture wasquenched with H₂O (100 mL) and transferred to a separatory funnel withEtOAc. The phases were separated and the aqueous phase was extractedwith EtOAc (100 mL×2) and DCM/MeOH (5:1, 100 mL×5). The combined organicextracts were concentrated under vacuum. The crude residue thus obtainedwas purified via flash column chromatography (240 g SiO₂, Isco, 9%MeOH/DCM to afford A-3 (19.7 g, 88%) as a yellow solid. LCMS [M+H]=322observed; ¹H NMR (400 MHz, DMSO-d6) δ ppm=11.18 (s, 1H), 8.79-8.73 (m,1H), 8.45-8.38 (m, 1H), 8.05 (br d, J=7.5 Hz, 2H), 7.73-7.51 (m, 4H),6.30-6.20 (m, 1H), 6.07 (br d, J=5.4 Hz, 1H), 5.61 (br dd, J=3.9, 5.2Hz, 1H), 5.38 (d, J=6.2 Hz, 1H), 4.81-4.72 (m, 1H), 3.02-2.91 (m, 1H),1.79 (td, J=4.5, 13.8 Hz, 1H).

Step 2: Synthesis ofN-(9-((1R,4S)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)cyclopent-2-en-1-yl)-9H-purin-6-yl)benzamide(A-4)

To a round bottom flask, equipped with a magnetic stirbar, was added A-3(19.7 g, 61.3 mmol) and anhydrous pyridine (50 mL). The solution wasconcentrated to dryness under vacuum and further dried under high vacuumfor 1 h. The flask was purged with N₂ followed by the addition ofanhydrous pyridine (150 mL) and DMTCl (23.9 g, 70.5 mmol). The reactionwas stirred at 9° C. under N₂ for 12 hours. The reaction was quenchedwith H₂O and transferred to a separatory funnel with EtOAc. The phaseswere separated and the aqueous phase was extracted with EtOAc (200mL×2). The organic extracts were washed with brine (100 mL×2), dried(Na₂SO₄), filtered, and concentrated under vacuum. The crude residuethus obtained was purified via flash column chromatography (240 g SiO₂,Isco, 100% EtOAc) to afford A-4 (29.2 g, 76%) as a yellow solid. LCMS[M+H]=624 observed; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm=8.94 (br s,1H), 8.79 (s, 1H), 8.26 (s, 1H), 8.03 (br d, J=7.5 Hz, 2H), 7.64-7.59(m, 1H), 7.57-7.47 (m, 4H), 7.44-7.36 (m, 4H), 7.35-7.28 (m, 2H),7.26-7.20 (m, 1H), 6.87-6.81 (m, 4H), 5.90 (dd, J=1.5, 5.5 Hz, 1H), 5.65(td, J=1.8, 5.5 Hz, 1H), 5.56-5.48 (m, 1H), 4.78-4.71 (m, 1H), 3.80 (d,J=1.0 Hz, 6H), 2.58-2.47 (m, 1H), 1.56 (td, J=3.6, 14.7 Hz, 1H).

Step 3: Synthesis ofN-(9-((1R,2S,3S,4S)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-2,3-dihydroxycyclopentyl)-9H-purin-6-yl)benzamide(A-5)

To round bottom flask, equipped with a magnetic stirbar, was added A-4(29.2 g, 46.8 mmol), DCM (300 mL), and H₂O (18 mL). To the yellowsolution was added NMO (16.5 g, 140 mmol) and OsO₄ (4% in t-BuOH, 20.8g, 3.28 mmol). The reaction was stirred at 16° C. for 5 hours. Thereaction was then transferred to a separatory funnel with DCM (50 mL)and quenched with saturated Na₂SO₃ (100 mL). The phases were separatedand the organic phase was washed with brine (100 mL), dried (Na₂SO₄),filtered, and concentrated under vacuum. The crude residue thus obtainedwas purified via flash column chromatography (120 g SiO₂, Isco, 3%MeOH/DCM to 5% MeOH/DCM) to afford A-5 (24.9 g, 80%) as a yellow solid.LCMS [M+H]=658 observed; ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm=9.09 (brs, 1H), 8.78-8.59 (m, 1H), 8.02 (br d, J=7.3 Hz, 2H), 7.93-7.85 (m, 1H),7.60 (br dd, J=4.8, 6.3 Hz, 1H), 7.56-7.39 (m, 5H), 7.39-7.27 (m, 6H),7.25-7.19 (m, 1H), 6.84 (br d, J=8.8 Hz, 4H), 5.69 (br s, 1H), 4.65 (brd, J=7.0 Hz, 2H), 4.16 (br d, J=1.3 Hz, 1H), 3.92 (br s, 1H), 3.78 (d,J=2.0 Hz, 6H), 3.72 (t, J=4.5 Hz, 2H), 2.95 (br s, 1H), 2.45-2.33 (m,2H), 1.94-1.75 (m, 1H).

Step 4: Synthesis ofN-(9-((1R,2S,3S,4S)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyldimethylsilyl)oxy)-2-hydroxycyclopentyl)-9H-purin-6-yl)benzamide(A-6)

To an oven dried round bottom flask, equipped with a magnetic stirbarand cooled under N₂, was added A-5 (4.12 g, 6.264 mmol), DCM (200 mL),Et₃N (3170 mg, 31.3 mmol) and TBSOTf (2.49 mg, 9.4 mmol) at 0° C.dropwise. The ice bath was removed and the reaction was stirred under N₂at 20° C. for 12 hours. At this stage, starting material was stilldetected by LCMS and an additional aliquot of TBSOTf (2485 mg, 9.4 mmol)was added to the mixture at 0° C. dropwise. The ice bath was removed andthe reaction mixture was stirred under N₂ at 20° C. for 12 hours. Thereaction was quenched with MeOH (15 mL). The solution was transferred toa separatory funnel with DCM and further diluted with H₂O. The phaseswere separated and the organic phase was washed with 1 portion H₂O, 1portion brine, dried (Na₂SO₄), filtered, and concentrated under vacuum.The crude residue was purified via flash column chromatography (40 gSiO₂, Isco, 40% EtOAc/Pet. Ether to 50% EtOAc/Pet. Ether) to afford A-6(1.29 g, 26%) as a white solid. LCMS [M+H]=772 observed; ¹H NMR (400MHz, CHLOROFORM-d) δ ppm=8.95 (s, 1H), 8.78 (s, 1H), 8.09 (s, 1H),8.05-7.98 (m, 2H), 7.64-7.57 (m, 1H), 7.56-7.47 (m, 3H), 7.56-7.47 (m,1H), 7.44-7.34 (m, 4H), 7.33-7.28 (m, 2H), 7.26-7.19 (m, 1H), 6.88-6.79(m, 4H), 4.76 (dd, J=5.6, 11.7 Hz, 1H), 4.70-4.62 (m, 1H), 4.38 (br s,1H), 4.10-4.06 (m, 1H), 3.78 (d, J=1.3 Hz, 6H), 2.98 (d, J=8.8 Hz, 1H),2.17 (s, 1H), 0.99 (br dd, J=4.6, 15.2 Hz, 1H), 0.93-0.85 (m, 9H), 0.14(s, 3H), 0.03 (s, 3H).

Step 5: Synthesis of(1S,2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl(2-cyanoethyl) diisopropylphosphoramidite (A-7)

To an oven dried round bottom flask, equipped with a magnetic stirbarand purged with N₂, was added A-6 (8.80 g, 11.4 mmol), DIPEA (14.7 g,114 mmol), and anhydrous DCM (320 mL). To the solution was added3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (13.5 g, 57.0mmol) at 15° C. under N₂. The reaction was stirred at 15° C. for 4hours. The reaction was quenched with sat. NaHCO₃ (120 mL) andtransferred to a separatory funnel with DCM (50 mL). The phases wereseparated and the organic phase was washed with brine (100 mL), dried(Na₂SO₄), and concentrated under vacuum. The crude residue thus obtainedwas purified via flash column chromatography (120 g SiO₂, Isco, 40%EtOAc/Pet. Ether to 60% EtOAc/Pet. Ether) twice to afford A-7 (8.57 g,77%) as a light yellow solid. LCMS [M+H]=972 observed; ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm=8.91 (d, J=11.3 Hz, 1H), 8.79 (d, J=4.0 Hz, 1H),8.22-8.13 (m, 1H), 8.08-7.98 (m, 2H), 7.65-7.58 (m, 1H), 7.52 (q, J=7.2Hz, 4H), 7.44-7.35 (m, 4H), 7.31 (dt, J=1.4, 7.5 Hz, 2H), 7.25-7.21 (m,1H), 6.84 (td, J=2.0, 9.0 Hz, 4H), 5.15-5.00 (m, 1H), 4.99-4.79 (m, 1H),4.31-3.98 (m, 2H), 3.78 (s, 6H), 3.76-3.57 (m, 2H), 3.50-3.34 (m, 2H),2.60 (t, J=6.3 Hz, 1H), 2.52-2.25 (m, 2H), 1.33-1.14 (m, 1H), 1.11 (d,J=6.8 Hz, 3H), 1.02 (t, J=7.4 Hz, 6H), 0.85 (s, 9H), 0.79 (d, J=6.8 Hz,3H), 0.11 (d, J=11.8 Hz, 3H), −0.06 (d, J=0.8 Hz, 3H); ³¹P NMR (162 MHz,CHLOROFORM-d) δ ppm=149.67 (s, 1P), 147.94 (s, 1P).

Step 1: Synthesis ofN-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(B-2)

To a round bottom flask, equipped with a magnetic stirbar, was addedcommercially availableN-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamideB-1 (42.00 g, 37.50 mmol) and co-evaporated with anhydrous pyridinethree times. The residue was re-dissolved in pyridine (70 mL) followedby the addition of DMTCl (13.98 g, 41.25 mmol) at 0° C. The mixture wasstirred at 25° C. for 12 h. The reaction mixture was concentrated underreduced pressure. The residue was diluted with DCM (200 mL) and washedwith three (200 mL) portions of saturated aqueous NaHCO₃. The organiclayer was dried over Na₂SO₄ (25.00 g), filtered and concentrated underreduced pressure. The crude residue was purified by flash columnchromatography (SiO₂, 85% Pet. ether/EtOAc to 100% EtOAc) to afford B-2(68.7 g, 85%) as a white solid which was used in the next step withoutfurther purification. LCMS [M+H]=676 observed.

Step 2: Synthesis of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (B-3)

Note: Six reactions were carried out in parallel. To a round bottomflask, equipped with a magnetic stirbar, was added phosphonic acid (21.8g, 266 mmol) and co-evaporated with anhydrous pyridine (60 mL) threetimes. The residue was dissolved in anhydrous pyridine (180 mL) withmild heating (30° C.). To the solution was added B-2 (12.0 g, 17.8 mmol)at 30° C. after which the solution thus obtained was cooled to 0° C. Tothis mixture was added 2,2-dimethylpropanoyl chloride (21.4 g, 177 mmol)dropwise at 0° C. The mixture was warmed to 30° C. and stirred for 12 h.The six reactions were combined. The reaction mixture was quenched with1M TEAB (1200 mL) and transferred to a separatory funnel. The solutionwas extracted with three (1000 mL) portions EtOAc. The combined organicextracts were washed with 0.5M TEAB (500 mL), brine (500 mL), dried overNa₂SO₄ (35.0 g), filtered and concentrated under vacuum. The cruderesidue was purified by flash column chromatography (SiO₂, 2% MeOH/DCMto 5% MeOH/DCM, 1% TEA) to remove major impurities and afford B-3 (100.0g, crude) as a white solid which was used in the next step withoutfurther purification. LCMS [M−H]=738 observed.

Step 3: Synthesis of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ylhydrogen phosphonate (B-4)

Note: Five reactions were carried out in parallel. To a round bottomflask, equipped with a magnetic stir bar, was added B-3 (20.0 g, 27.0mmol) and a solution of DCA (17.4 g, 135 mmol) in DCM (200 mL) and wasadded. The mixture was stirred at 25° C. for 0.5 h. The five reactionswere combined. The solid product in reaction mixture was filtered andtriturated with DCM (300 mL) to afford B-4 (40.0 g, 63%) as a whitesolid. LCMS [M+H]=438 observed; ¹H NMR (400 MHz, DMSO-d6) δppm=11.95-10.65 (m, 1H), 8.78 (s, 1H), 8.73 (s, 1H), 8.05 (d, J=7.3 Hz,2H), 7.71-7.61 (m, 1H), 7.61-7.49 (m, 2H), 6.69 (s, 1H), 6.46 (dd,J=3.1, 16.6 Hz, 1H), 5.82 (td, J=3.4, 51.7 Hz, 1H), 5.30-5.16 (m, 1H),4.31-4.20 (m, 1H), 3.80 (dd, J=2.8, 12.2 Hz, 1H), 3.67 (dd, J=3.7, 12.5Hz, 1H); ¹⁹F NMR (377 MHz, DMSO-d6) δ ppm=−203.02 (s, 1F); ³¹P NMR (162MHz, DMSO-d6) δ ppm=4.20 (s, 1P).

Step 1: Synthesis of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((1S,2R,3S,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((tert-butyldimethylsilyl)oxy)-3-hydroxycyclopentyl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (C-1)

To a round bottom flask, equipped with a magnetic stirbar, was added theH-phosphonate B-4 (1.0 g, 2.29 mmol) and pyridinium trifluoroacetate(1.77 g, 9.15 mmol). The solids were taken up in anhydrous MeCN (10mL×2) and concentrated under vacuum. The residue was re-dissolved inanhydrous MeCN (30 mL) and 3 A molecular sieves (4.0 g) were added. Thesolution was stirred for 30 minutes at which point phosphoramidite A-7(2.67 g, 2.74 mmol) was added. The reaction was stirred at rt for 1.5hours during which a homogeneous solution was obtained. To the reactionwas added DDTT (493 mg, 2.40 mmol) and the reaction was allowed to stirat rt overnight. The reaction was filtered and the solids washed withMeOH/DCM (1:1). The filtrate was concentrated under vacuum followed bytrituration with n-hexane/TBME (1:1). The crude residue thus obtainedwas purified via preparatory high performance liquid chromatography(Phenomenex Synergi Max-RP 150×50 mm×10 μm, 20% MeCN/H₂O with 10 mMNH₄HCO₃ to 50% MeCN/H₂O with 10 mM NH₄HCO₃, 120 mL/min) to afford C-1(390 mg, 13%) as a white solid. LCMS [M+H]=1038 observed; ³¹P NMR (162MHz, METHANOL-d4) δ ppm=68.24 (s, 1P), 67.74 (s, 1P), 2.95 (s, 1P), 2.71(s, 1P).

Step 2: Synthesis ofN,N′-{[(4S,6R,7S,11aR,13R,14R,14aR,15R)-15-{[tert-butyl(dimethyl)sily]oxy}-9-(2-cyanoethoxy)-14-fluoro-2-oxido-2-sulfanyl-9-sulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecine-6,13-diyl]bis(9H-purine-9,6-diyl)}dibenzamide(C-2)

To an oven dried round bottom flask, equipped with a magnetic stirbarand cooled under N₂, was added C-1 (320 mg, 0.308 mmol) and anhydrouspyridine (10 mL). To the solution was added DMOCP (1.02 g, 5.55 mmol) atrt under N₂. The reaction was stirred under N₂ for 1 hour at which pointthe starting material had been consumed. To the reaction was added H₂O(333 mg, 18.5 mmol) and 3H-1,2-benzodithiol-3-one (130 mg, 0.770 mmol)at rt under N₂. The reaction was stirred under N₂ at rt for 30 minutes.The reaction was quenched with sat. NaHCO₃ and transferred to aseparatory funnel with EtOAc. The phases were separated and the aqueousphase was extracted with 2 portions EtOAc (60 mL). The combined organicextracts were washed with brine, dried (Na₂SO₄), filtered andconcentrated under vacuum. The crude residue was purified via flashcolumn chromatography (SiO₂, Isco, 9% MeOH/DCM to 11% MeOH/DCM). Thematerial obtained was further purified via preparatory thin layerchromatography (SiO₂, 11% MeOH/DCM) to afford C-2 (135 mg, 41%) as awhite solid. LCMS [M+H]=1052 observed; ³¹P NMR (162 MHz, METHANOL-d4) δppm=68.90 (s, 1P), 68.33 (s, 1P), 64.75 (s, 1P), 64.68 (s, 1P), 55.42(s, 1P), 53.26 (s, 1P), 52.98 (s, 1P).

Step 3: Synthesis ofN,N′-{[4S,6R,7S,11aR,13R,14R,14aR,15R)-15-{[tert-butyl(dimethyl)silyl]oxy}-14-fluoro-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecine-6,13-diyl]bis(9H-purine-9,6-diyl)}dibenzamide(C-3)

To a flask containing C-2 (130 mg, 0.124 mmol) was added acetonitrile (9mL). To the resulting suspension was added 4.5 mL tert-butylamine. Thereaction mixture became a solution and was stirred at room temperaturefor 20 min. The reaction was concentrated to give crude C-3 as whitesolid, used for next step.

LCMS [M+H]=999.21

³¹P NMR (162 MHz, METHANOL-d4) δ ppm 57.61 (s, 1P) 57.20 (s, 1P) 55.15(s, 1P) 54.77 (s, 1P) 54.00 (s, 1P) 53.94 (s, 1P) 52.30 (s, 1P) 52.04(s, 1P)

Step 4: Synthesis of9,9′-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-15-{[tert-butyl(dimethyl)silyl]oxy}-14-fluoro-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecine-6,13-diyl]bis(9H-purin-6-amine)(C-4)

The crude material C-3 (123 mg, 0.123 mmol) was added to 6 mL of 33%methylamine in EtOH (0.02 M). The solution stirred at room temperatureovernight. The reaction mixture was concentrated to give crude C-4 thatwas used for the next step.

LCMS [M+H]=791.15

³¹P NMR (162 MHz, METHANOL-d4) δ ppm 58.63 (br. s., 1P) 55.08 (br. s.,1P) 54.57 (br. s., 1P) 52.50 (s, 1P) 52.26 (s, 1P)

¹⁹F NMR (376 MHz, METHANOL-d4) δ ppm −200.04 (s, 1F) −200.59 (s, 1F)−201.31 (br. s., 1F)

Step 5: Synthesis of(4S,6R,7S,11aR,13R,14R,14aR,15R)-6,13-bis(6-amino-9H-purin-9-yl)-14-fluoro-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-15-ol2,9-dioxide (C-5)

The material C-4 (97.4 mg, 0.123 mmol) was co-evaporated with 4 mLpyridine/Et₃N (v/v, 3/1) three times. The material was dissolved in 1.13mL pyridine (0.1 M), charged with N₂, triethylamine (0.102 mL, c=0.1 M)and triethylamine trihydrofluoride (993 mg, 6.16 mmol) were added. Theresulting reaction mixture was heated at 50° C. for overnight. Thereaction mixture was quenched with NaHCO₃ solution to pH 6. The volatilecomponents were removed in vacuo. The residue was purified by reversephase prep-HPLC (Phenomenex Gemini C18 21.2×150 mm 5 u column) elutedwith 10-40% MeCN in aq. NH₄HCO₃ (10 mM) to give two diastereomericcompounds as white solid, and a mixture of the other diastereomers. Theother two diastereomers were separated on Phenomenex Luna Omega 5 uPolar C18 21.2×150 mm column eluted with 8-40% MeCN in aq. NH₄HCO₃ (10mM).

Peak 1, 13.76 mg, 15.8%;

LCMS [M+H]=677.00

¹H NMR (400 MHz, DEUTERIUM OXIDE) δ ppm 8.42 (s, 1H) 8.34 (s, 1H) 8.30(s, 1H) 7.90 (s, 1H) 6.49 (d, J=16.51 Hz, 1H) 6.27 (dd, J=49.34, 4.28Hz, 1H) 5.36-5.46 (m, 1H) 5.10-5.24 (m, 2H) 4.65-4.69 (m, 1H) 4.62 (br.s., 1H) 4.54 (d, J=8.93 Hz, 1H) 4.34 (d, J=12.84 Hz, 1H) 4.15 (dd,J=12.29, 5.69 Hz, 1H) 2.99-3.11 (m, 1H) 2.28 (dd, J=15.96, 6.05 Hz, 1H)

³¹P NMR (162 MHz, DEUTERIUM OXIDE) δ ppm 56.18 (br. s., 1P) 50.55 (s,1P)

¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ ppm −200.13 (s, 1F)

Peak 2, 9.97 mg, 9%;

LCMS [M+H]=677.00

¹H NMR (400 MHz, DEUTERIUM OXIDE) δ ppm 8.39 (s, 1H) 8.27 (s, 1H) 8.27(s, 1H) 7.85 (s, 1H) 6.47 (d, J=16.63 Hz, 1H) 5.74 (m, 1H) 5.32-5.42 (m,1H) 5.11-5.27 (m, 2H) 4.68-4.73 (m, 1H) 4.60 (br. s., 1H) 4.51 (d,J=9.05 Hz, 1H) 4.31 (d, J=11.98 Hz, 1H) 4.07-4.15 (m, 1H) 3.02-3.11 (m,1H) 2.31 (dd, J=15.96, 6.42 Hz, 1H)

³¹P NMR (162 MHz, DEUTERIUM OXIDE) δ ppm 57.60 (s, 1P) 53.47 (s, 1P)

¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ ppm −199.70 (s, 1F)

Peak 3, 15.25 mg, 17.5%;

LCMS [M+H]=677.00

¹H NMR (400 MHz, DEUTERIUM OXIDE) δ ppm 8.27 (s, 1H) 8.26 (s, 1H) 8.22(s, 1H) 7.73 (s, 1H) 6.45 (d, J=16.75 Hz, 1H) 6.27 (dd, J=50.25, 3.79Hz, 1H) 5.46-5.53 (m, 1H) 5.04-5.18 (m, 2H) 4.93-5.09 (m, 1H) 4.61-4.67(m, 2H) 4.55 (d, J=9.66 Hz, 1H) 4.41 (d, J=12.23 Hz, 1H) 4.06-4.14 (m,1H) 2.94-3.09 (m, 1H) 2.20 (dd, J=15.47, 6.30 Hz, 1H)

³¹P NMR (162 MHz, DEUTERIUM OXIDE) δ ppm 51.67 (br. s., 1P) 50.40 (s,1P)

¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ ppm −200.23 (s, 1F)

Peak 4, 5.73 mg, 6.6%;

LCMS (ES, m/z): 677.00 [M+H]

¹H NMR (400 MHz, DEUTERIUM OXIDE) δ ppm 8.26 (s, 1H) 8.24 (s, 1H) 8.21(s, 1H) 7.74 (s, 1H) 6.44 (d, J=17.24 Hz, 1H) 5.73 (m., 1H) 5.41-5.51(m, 1H) 5.05-5.20 (m, 2H) 4.70 (m, 1H) 4.61 (br. s., 1H) 4.51 (d, J=8.19Hz, 1H) 4.38 (d, J=11.86 Hz, 1H) 4.01-4.16 (m, 1H) 3.02 (m, 1H)2.15-2.29 (m, 1H)

³¹P NMR (162 MHz, DEUTERIUM OXIDE) δ ppm 51.91 (s, 1P) 51.56 (s, 1P)

¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ ppm −199.85 (s, 1F)

Example 29-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one

Step 1: Synthesis of(1S,4R)-4-(6-chloro-9H-purin-9-yl)cyclopent-2-en-1-ol (D-2)

A mixture of A-1 (1500 mg, 11 mmol), Pd(PPh₃)₄ (610 mg, 0.53 mmol), andPPh₃ (277 mg, 1.06 mmol), in THF (6 mL) was bubbled with N₂ for 15 min(flask A). In a separate flask (flask B), a suspension of D-1 in amixture of THF (30 mL) and DMA (5 mL) was bubbled with N₂ for 15 minthen NaH (60 wt % dispersion in mineral oil, 506 mg, 12.7 mmol) wasadded. After 1.5 hr, the contents of flask A were transferred to flask B(rinsed with 4 mL THF) and the reaction was heated at 50° C. overnight.The reaction mixture was concentrated; the residue was dissolved inEtOAc and washed with aq citric acid and brine. The combined aqueousphases were extracted with EtOAc (2×). The combined organic phases weredried over MgSO₄, filtered and concentrated. The crude residue waspurified via flash chromatography (40 g SiO₂, Isco, 0-5% MeOH/DCM) toafford D-2 (1.45 g, 58%) as a foamy yellow solid. LCMS [M+H]=237observed; ¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.79 (s, 1H) 8.60 (s, 1H) 6.24(dt, J=5.53, 1.94 Hz, 1H) 6.00-6.11 (m, 1H) 5.60 (td, J=5.14, 1.96 Hz,1H) 5.30 (d, J=6.24 Hz, 1H) 4.65-4.81 (m, 1H) 2.94 (ddd, J=14.00, 8.13,7.34 Hz, 1H) 1.80 (dt, J=14.03, 4.48 Hz, 1H).

Step 2: Synthesis of9-[(1R,4S)-4-hydroxycyclopent-2-en-1-yl]-1,9-dihydro-6H-purin-6-one(D-3)

To a solution of D-2 (1.45 g, 7.03 mmol) in MeOH (50 mL) was addedmercaptoethanol (1.97 mL, 28.1 mmol) followed by sodium methoxide (1.52g, 28.1 mmol). After refluxing for 8 hrs, the heat was turned off andthe reaction was left standing overnight. The reaction was concentratedand used directly in the next step. LCMS [M+H]=219 observed.

Step 3: Synthesis of9-[(1R,4S)-4-hydroxycyclopent-2-en-1-yl]-1-methyl-1,9-dihydro-6H-purin-6-one(D-4)

To a solution of D-3 (used crude from previous step) in DMF (47 mL) wasadded K₂CO₃ (3.39 g, 24.5 mmol) followed by methyl iodide (0.655 mL,10.5 mmol). After stirring overnight, another 2 eq methyl iodide (0.873mL, 14.0 mmol) was added. After 1 hr, the reaction was concentrated. Theresidue was slurried in DCM and filtered to remove solids. The motherliquor was concentrated and purified via flash chromatography (40 gSiO₂, Isco, 0-10% 7N NH₃ in MeOH/DCM) to afford D-4 (1.17 g, 72%) as awhite solid. LCMS [M+H]=233 observed; ¹H NMR (400 MHz, DMSO-d₆) δppm=8.39 (s, 1H), 8.00 (s, 1H), 6.19 (td, J=2.0, 5.5 Hz, 1H), 6.05-5.90(m, 1H), 5.41 (dt, J=2.0, 5.2 Hz, 1H), 5.26 (d, J=6.4 Hz, 1H), 4.76-4.66(m, 1H), 3.50 (s, 3H), 2.89 (ddd, J=7.3, 8.2, 13.9 Hz, 1H), 1.68 (td,J=4.5, 13.9 Hz, 1H).

Step 4: Synthesis of9-{(1R,4S)-4-[bis(4-methoxyphenyl)(phenyl)methoxy]cyclopent-2-en-1-yl}-1-methyl-1,9-dihydro-6H-purin-6-one(D-5)

The compound D-4 (1.17 g, 5.04 mmol) was co-evaporated with anhydrouspyridine (3×). Dissolved residue a final time in anhydrous pyridine (34mL), added DMTCl (1.96 g, 1.15 mmol) and stirred overnight. The pyridinewas removed in vacuo then the residue was dissolved in EtOAc and washedwith water and brine. The organics were dried over MgSO₄, filtered andconcentrated. The crude residue was purified via flash chromatography(80 g SiO₂, Isco, 0-5% MeOH/DCM) to afford D-5 (2.50 g, 93%) as a yellowsolid. LCMS [M+H]=535 observed; ¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.36 (s,1H) 7.98 (s, 1H) 7.44 (d, J=8.07 Hz, 2H) 7.28-7.36 (m, 6H) 7.19-7.29 (m,1H) 6.91 (d, J=8.31 Hz, 4H) 5.97 (d, J=5.62 Hz, 1H) 5.46 (d, J=5.50 Hz,1H) 5.24 (br. s., 1H) 4.60 (br. s., 1H) 3.74 (s, 6H) 3.50 (s, 3H)2.34-2.45 (m, 1H) 1.54 (dt, J=13.72, 4.69 Hz, 1H).

Step 5: Synthesis of9-{(1R,2S,3S,4S)-4-[bis(4-methoxyphenyl)(phenyl)methoxy]-2,3-dihydroxycyclopentyl}-1-methyl-1,9-dihydro-6H-purin-6-one(D-6)

To a solution of D-5 (2.50 g, 4.68 mmol) in DCM (31.2 mL) was addedwater (3.12 mL), NMMO (1.64 g, 14.0 mml), and OsO₄ (2.5 wt % in^(t)BuOH, 3.33 mL, 0.327 mmol). After stirring overnight the reactionwas diluted with EtOAc then washed with sat'd Na₂SO₃ and brine. Theorganics were dried over MgSO₄, filtered and concentrated. The cruderesidue was purified via flash chromatography (80 g SiO₂, Isco, 0-8%MeOH/DCM) to afford D-6 (2.37 g, 89%). LCMS [M+H]=569 observed; ¹H NMR(400 MHz, DMSO-d₆) δ ppm=8.34 (s, 1H) 8.06 (s, 1H) 7.38-7.49 (m, 2H)7.27-7.36 (m, 6H) 7.19-7.26 (m, 1H) 6.89 (dd, J=9.05, 2.32 Hz, 4H) 5.01(d, J=6.11 Hz, 1H) 4.74 (d, J=3.79 Hz, 1H) 4.41-4.58 (m, 2H) 3.80-3.87(m, 1H) 3.73 (d, J=2.20 Hz, 6H) 3.56-3.62 (m, 1H) 3.50 (s, 3H) 1.84-1.97(m, 1H) 1.36-1.55 (m, 1H).

Step 6: Synthesis of9-[(1R,2S,3S,4S)-4-[bis(4-methoxyphenyl)(phenyl)methoxy]-3-{[tert-butyl(dimethyl)silyl]oxy}-2-hydroxycyclopentyl]-1-methyl-1,9-dihydro-6H-purin-6-one(D-7)

The compound D-6 (1.88 g, 3.31 mmol) was co-evaporated with anhydrouspyridine (3×). Dissolved residue a DMF (33 mL), added imidazole (682 mg,9.92 mmol) then TBSCl (747 mg, 4.96 mmol) and stirred overnight. The DMFwas removed in vacuo then the residue was dissolved in EtOAc and washedwith water and brine. The organics were dried over MgSO₄, filtered andconcentrated. The crude residue was purified via flash chromatography(80 g SiO₂, Isco, 0-100% EtOAc/heptanes) to afford D-7 (793 mg, 35%) asa white solid. LCMS [M+H]=683 observed; ¹H NMR (400 MHz, DMSO-d₆) δppm=8.34 (s, 1H) 8.01 (s, 1H) 7.42-7.49 (m, 2H) 7.28-7.37 (m, 6H)7.20-7.27 (m, 1H) 6.85-6.93 (m, 4H) 5.12 (d, J=5.62 Hz, 1H) 4.63-4.71(m, 1H) 4.46-4.56 (m, 1H) 3.78-3.84 (m, 2H) 3.73 (d, J=1.71 Hz, 6H) 3.51(s, 3H) 2.08 (ddd, J=14.58, 10.30, 6.05 Hz, 1H) 1.29 (dd, J=14.37, 6.79Hz, 1H) 0.82 (s, 9H) 0.04 (s, 3H) −0.06 (s, 3H)

Step 7: Synthesis of(1S,2R,3S,5R)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl2-cyanoethyl dipropan-2-ylphosphoramidoite (D-8)

To a solution of D-7 (785 g, 1.15 mmol) in DCM (23 mL) was added DIEA(601 mL, 3.45 mmol) followed by3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (385 uL, 1.72mmol) drop-wise. After 1 hour, added an additional 0.75 eq3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile drop-wise.After another hour, the reaction was diluted with EtOAc and washed withsaturated NaHCO₃ and brine. The organics were dried over MgSO₄, filteredand concentrated. The crude residue was purified via flashchromatography (40 g SiO₂, Isco, 0-100% EtOAc/heptanes) to afford D-8(779 mg, 77%) as a white solid. LCMS [M+H]=800 observed; ¹H NMR (400MHz, DMSO-d₆) δ ppm=8.36 (d, J=1.83 Hz, 1H) 7.95 (d, J=14.18 Hz, 1H)7.48 (d, J=7.95 Hz, 2H) 7.33 (td, J=5.84, 2.51 Hz, 6H) 7.22-7.29 (m, 1H)6.87-6.95 (m, 4H) 4.69-4.92 (m, 2H) 3.85 (d, J=6.36 Hz, 1H) 3.74 (s, 6H)3.70 (br. s., 1H) 3.56-3.67 (m, 1H) 3.51 (d, J=6.24 Hz, 3H) 3.33-3.48(m, 3H) 2.54-2.76 (m, 2H) 2.09-2.48 (m, 1H) 1.21-1.53 (m, 1H) 0.95-1.08(m, 9H) 0.71-0.85 (m, 12H) 0.06 (d, J=19.32 Hz, 3H) −0.11 (d, J=10.51Hz, 3H); ³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄) δppm=148.32 (s, 1P), 146.67 (s, 1P).

Step 1: Synthesis ofN-benzoyl-5′-O-[{[(1S,2R,3S,5R)-2-{[tert-butyl(dimethyl)silyl]oxy}-3-hydroxy-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl]oxy}(2-cyanoethoxy)phosphorothioyl]-2′-deoxy-2′-fluoro-3′-O-[hydroxy(oxido)-I⁵-phosphanyl]adenosine(E-1)

The compound D-8 (709 mg, 0.803 mmol) was co-evaporated with THF (3×).Dissolved a final time in THF (20 mL) added powdered molecular sievesand stirred for 1 hour (flask A). A mixture of B-4 (351 mg, 0.803 mmol)and pyTFA (930 mg, 4.82 mol) was co-evaporated with THF (3×). Dissolveda final time in THF (20 mL) added powdered molecular sieves and stirredfor 1 hour (flask B). The contents of flask B were then added to flaskA. After 30 min, DDTT (330 mg, 1.61 mmol) was added. After another 30min, the reaction mixture was concentrated and the residue was slurriedin DCM. Filtered out molecular sieves and concentrated mother liquor.Dissolved residue in DCM (4 mL) added a few drops of water then asolution of DCA (662 uL, 8.03 mmol) in DCM (4 mL) resulting in a brightorange solution. After 30 min, pyridine was added until the orange colordissipated. The reaction mixture was concentrated and purified via flashchromatography (40 g SiO₂, Isco, 0-40% MeOH/DCM then 12 g SiO₂, Isco,0-40% MeOH/DCM) to afford E-1 (227 mg, 30%). LCMS [M+H]=950 observed;³¹P NMR (162 MHz, DMSO-d₆) δ ppm=67.93 (s., 1P), 65.55 (s, 1P), −0.34(s, 1P), −0.68 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−200.59 (s,1F), −201.37 (s, 1F).

Step 2: Synthesis ofN-{9-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-15-{[tert-butyl(dimethyl)silyl]oxy}-9-(2-cyanoethoxy)-14-fluoro-6-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-oxido-2-sulfanyl-9-sulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-13-yl]-9H-purin-6-yl}benzamide(E-2)

The compound E-1 (227 mg, 0.239 mmol) was co-evaporated with anhydrouspyridine (3×). Dissolved residue a final time in anhydrous pyridine (12mL) then added DMOCP (221 mg, 1.20 mmol). After 1 hour an additional 5eq of DMOCP were added. After 4 hours, added water (1.0 mL) followed by3H-1,2-benzodithiol-3-one (81 mg, 0.48 mmol). After 30 min the reactionwas quenched with saturated NaHCO₃, concentrated and purified via flashchromatography (24 g SiO₂, Isco, 0-40% MeOH/DCM) to afford E-2 (155 mg,67%). LCMS 4 peaks with [M+H]=964 observed; ³¹P NMR (162 MHz, DMSO-d₆) δppm=67.40 (s, 1P), 67.02 (s, 1P), 63.92 (s, 1P), 63.80 (s, 1P), 49.90(s, 1P), 49.83 (s, 1P), 49.38 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d₆) δppm=−195.70 (s, 1F), −196.27 (s, 1F), −196.54 (s, 1F), −196.55 (s, 1F)

Step 3: Synthesis of9-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one(E-3)

To a solution of E-2 (260 mg, 0.270 mmol) in ACN (6 mL) was added^(t)BuNH₂. After 15 min, the reaction was concentrated and the residuewas dissolved in 33% MeNH₂ in EtOH (6 mL). After 2 hrs, the reaction wasconcentrated and the residue was co-evaporated with 3:1 pyridine:TEA(2×). Dissolved the residue in pyridine (3 mL), added TEA (300 uL)followed by triethylamine trihydrofluoride (2.2 mL, 13.5 mmol) andheated to 50° C. overnight. Concentrated then adjusted pH to ˜6 withsaturated NaHCO₃. Concentrated, slurried residue in DCM, filtered outsolids then concentrated mother liquor. The residue was purified byreverse phase prep-HPLC (Phenomenex Gemini C18 21.2×150 mm 5 u column)eluted with 5-10% MeCN in aq. NH₄HCO₃ (10 mM) to give 4 diastereomers.Peaks 3 and 4 were re-purified using the same conditions.

Peak 1:

25 mg, 13%;

¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.44 (s, 1H) 8.21 (s, 1H) 8.20 (s, 1H)8.10 (s, 1H) 7.40 (br. s., 2H) 6.29 (d, J=17.85 Hz, 2H) 5.43-5.60 (m,1H) 5.07-5.16 (m, 1H) 4.97 (td, J=9.93, 5.93 Hz, 2H) 4.51-4.61 (m, 2H)4.23 (d, J=8.56 Hz, 1H) 4.00 (br. s., 2H) 3.50 (s, 3H) 2.83 (br. s., 1H)1.66 (dd, J=14.49, 5.81 Hz, 1H);

³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄) δ ppm=53.68 (s,1P), 50.53 (s, 1P);

¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−197.42 (s, 1F).

Peak 2:

32 mg, 16%;

¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.44 (s, 1H) 8.22 (s, 1H) 8.18 (s, 1H)8.13 (s, 1H) 7.40 (br. s., 2H) 6.28 (d, J=17.24 Hz, 1H) 6.02-6.20 (m,1H) 5.06-5.15 (m, 1H) 4.95 (td, J=9.90, 5.50 Hz, 2H) 4.56 (br. s., 1H)4.43 (t, J=5.26 Hz, 1H) 4.24 (d, J=8.44 Hz, 1H) 3.96-4.12 (m, 2H) 3.49(s, 3H) 2.80 (br. s., 1H) 1.61 (dd, J=14.92, 5.50 Hz, 1H);

³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄) δ ppm=53.33 (s,1P), 48.21 (s, 1P);

¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−198.33 (s, 1F).

Peak 3:

34 mg, 16%;

¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.41 (s, 1H) 8.36 (br. s., 1H) 8.23 (s,1H) 8.18 (s, 1H) 7.40 (br. s., 2H) 6.29 (d, J=16.38 Hz, 1H) 6.04-6.22(m, 1H) 5.12-5.26 (m, 1H) 4.77-4.96 (m, 2H) 4.37-4.48 (m, 2H) 4.28 (d,J=8.80 Hz, 1H) 4.18 (d, J=11.98 Hz, 1H) 4.02 (dd, J=11.74, 5.50 Hz, 1H)3.50 (s, 3H) 2.86 (br. s., 1H) 1.52 (dd, J=15.22, 5.81 Hz, 1H);

³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄) δ ppm=49.42 (s,1P), 48.13 (s, 1P);

¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−199.27 (s, 1F).

Peak 4:

7 mg, 3.5%;

¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.40 (s, 1H) 8.27 (s, 1H) 8.20 (s, 1H)8.19 (s, 1H) 7.38 (br. s., 2H) 6.27 (d, J=16.63 Hz, 1H) 5.40-5.60 (m,1H) 5.24 (d, J=3.42 Hz, 1H) 5.15-5.23 (m, 1H) 4.82-4.99 (m, 2H) 4.52 (t,J=6.36 Hz, 1H) 4.45 (br. s., 1H) 4.26 (d, J=9.29 Hz, 1H) 4.09-4.18 (m,1H) 3.97-4.06 (m, 1H) 3.49 (s, 3H) 2.85 (t, J=16.08 Hz, 1H) 1.56 (dd,J=14.67, 5.99 Hz, 1H);

³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄) δ ppm=50.54 (s,1P), 48.91 (s, 1P);

¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−198.50 (s, 1F).

Example 39-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1,9-dihydro-6H-purin-6-one

Step 1: Synthesis of(1S,4R)-4-(6-(benzyloxy)-9H-purin-9-yl)cyclopent-2-en-1-ol (F-2)

Compound F-2 was made in a similar fashion as A-3 using6-(benzyloxy)-9H-purine (F-1) in place of A-2 in step 1 of Scheme A in67% yield.

¹H NMR (400 MHz, DMSO-d₆) δ=8.56 (s, 1H), 8.32 (s, 1H), 7.55-7.48 (m,2H), 7.45-7.31 (m, 3H), 6.21 (td, J=2.0, 5.5 Hz, 1H), 6.09-5.99 (m, 1H),5.63 (s, 2H), 5.55 (dt, J=2.0, 5.2 Hz, 1H), 5.36 (d, J=6.4 Hz, 1H), 4.74(td, J=1.5, 3.1 Hz, 1H), 3.00-2.84 (m, 1H), 1.75 (td, J=4.4, 13.9 Hz,1H); LCMS [M+H]=309.0.

Step 2: Synthesis of6-(benzyloxy)-9-((1R,4S)-4-(bis(4-methoxyphenyl)(phenyl)-I4-oxidaneyl)cyclopent-2-en-1-yl)-9H-purine(F-3)

Compound F-3 was made in a similar fashion as A-4 in step 2 of Scheme Ain 82% yield.

¹H NMR (400 MHz, DMSO-d₆) δ=8.54 (s, 1H), 8.29 (s, 1H), 7.51 (d, J=7.0Hz, 2H), 7.47-7.35 (m, 5H), 7.35-7.28 (m, 6H), 7.26-7.20 (m, 1H), 6.90(dd, J=2.1, 9.0 Hz, 4H), 6.00 (d, J=6.0 Hz, 1H), 5.63 (s, 2H), 5.48-5.42(m, 1H), 5.38 (t, J=6.1 Hz, 1H), 4.61 (t, J=5.0 Hz, 1H), 3.73 (d, J=1.3Hz, 6H), 2.48-2.39 (m, 1H), 1.61 (td, J=4.8, 13.8 Hz, 1H); LCMS[M+H]=610.8.

Step 3: Synthesis of(1S,2S,3R,5S)-3-(6-(benzyloxy)-9H-purin-9-yl)-5-(bis(4-methoxyphenyl)(phenyl)-I4-oxidaneyl)cyclopentane-1,2-diol(F-4)

Compound F-4 was made in a similar fashion as A-5 in step 3 of Scheme Ain 76% yield.

¹H NMR (400 MHz, DMSO-d₆) δ=8.55 (s, 1H), 8.37 (s, 1H), 7.53-7.44 (m,4H), 7.40 (d, J=7.3 Hz, 3H), 7.36-7.27 (m, 6H), 7.26-7.18 (m, 1H), 6.88(dd, J=3.1, 8.9 Hz, 4H), 5.63 (s, 2H), 5.04 (d, J=6.1 Hz, 1H), 4.77 (d,J=3.7 Hz, 1H), 4.68-4.55 (m, 2H), 3.86 (td, J=2.4, 4.6 Hz, 1H), 3.72 (d,J=2.8 Hz, 6H), 3.59 (br. s., 1H), 2.01-1.90 (m, 1H), 1.66-1.55 (m, 1H);LCMS [M+H]=645.8.

Step 4: Synthesis of(1S,2S,3S,5R)-5-(6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)-I4-oxidaneyl)-2-((tert-butyldimethylsilyl)oxy)cyclopentan-1-ol(F-5)

Compound F-5 was made in a similar fashion as A-6 in step 3 of Scheme Ain 35% yield.

¹H NMR (400 MHz, DMSO-d₆) δ=8.56 (s, 1H), 8.32 (s, 1H), 7.49 (dd, J=7.2,14.1 Hz, 4H), 7.40 (d, J=7.3 Hz, 3H), 7.36-7.28 (m, 6H), 7.26-7.19 (m,1H), 6.88 (dd, J=3.2, 8.9 Hz, 4H), 5.64 (s, 2H), 5.14 (d, J=5.5 Hz, 1H),4.86-4.76 (m, 1H), 4.71-4.57 (m, 1H), 3.87-3.75 (m, 2H), 3.72 (d, J=2.3Hz, 6H), 2.13 (ddd, J=6.4, 10.3, 14.7 Hz, 1H), 1.44 (dd, J=6.9, 14.4 Hz,1H), 0.82 (s, 9H), 0.04 (s, 3H), −0.07 (s, 3H); LCMS [M+H]=758.8.

Step 5: Synthesis of(1S,2R,3S,5R)-5-(6-(benzyloxy)-9H-purin-9-yl)-3-(bis(4-methoxyphenyl)(phenyl)-I4-oxidaneyl)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl(2-cyanoethyl) diisopropylphosphoramidite (F-6)

Compound F-6 was made in a similar fashion as A-7 in step 3 of Scheme Ain 73% yield. ³¹P NMR (162 MHz, DMSO-d₆) δ=149.11 (br. s., 1P), 147.26(s, 1P); LCMS [M+H]=959.0.

Step 1: Synthesis of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((1S,2R,3S,5R)-5-(6-(benzyloxy)-9H-purin-9-yl)-2-((tert-butyldimethylsilyl)oxy)-3-hydroxycyclopentyl)oxy)(2-cyanoethoxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-ylhydrogen phosphonate (G-1)

Compound G-1 was made in a similar fashion as E-1 in step 1 of Scheme Ein 42% yield.

¹⁹F NMR (376 MHz, DMSO-d₆) δ=−201.22 (s, 1F), −201.70 (s, 1F); ³¹P NMR(162 MHz, DMSO-d₆) δ=68.34 (s, 1P), 65.79 (s, 1P), −0.03 (s, 1P), −0.15(s, 1P); LCMS [M+H]=1025.

Step 2: Synthesis ofN-{9-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-6-[6-(benzyloxy)-9H-purin-9-yl]-15-{[tert-butyl(dimethyl)silyl]oxy}-9-(2-cyanoethoxy)-14-fluoro-2-oxido-2-sulfanyl-9-sulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-13-yl]-9H-purin-6-yl}benzamide(G-2)

Compound G-2 was made in a similar fashion as E-2 in step 2 of Scheme Ein 29% yield.

¹⁹F NMR (376 MHz, DMSO-d₆) δ=−194.72 (s, 1F), −196.58 (s, 1F), −196.67(s, 1F), −196.96 (s, 1F); ³¹P NMR (162 MHz, DMSO-d₆) δ=67.91 (s, 1P),67.60 (br. s., 1P), 63.96 (br. s., 1P), 63.78 (br. s., 1P), 51.01 (s,2P), 49.48 (s, 1P), 48.94 (s, 1P); LCMS [M+H]=1039.

Step 3: Synthesis of9-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1,9-dihydro-6H-purin-6-one(G-5)

Compound G-2 was treated in a similar fashion as compound E-2 in step 3of Scheme E to give G-3 followed by G-4. Finally, to a solution of G-4(156 mg, 0.18 mmol) in MeOH (3.00 mL, c=0.06 M) was added 3N HCl (300mg, 9 mmol, 3.00 mL, 3 M). Upon addition of HCl a white precipitateformed. The suspension was heated to 50° C. During heating the reactionbecame a homogeneous yellow solution. Stirring was continued at 50° C.for 4.5 h. The reaction was cooled to rt then neutralized to pH 6 withNaHCO₃(sat). The aqueous mixture was lyophilized then purified by PrepHPLC to give 4 diastereomers.

Peak 1: 41 mgs, 28% yield, 95% de, ¹H NMR (400 MHz, DEUTERIUM OXIDE)δ=8.43 (s, 1H), 8.26 (s, 1H), 8.21 (s, 1H), 7.68 (s, 1H), 6.49 (d,J=16.1 Hz, 1H), 5.73-5.57 (m, 1H), 5.52-5.42 (m, 1H), 5.24-5.09 (m, 2H),4.60 (br. s., 1H), 4.52 (d, J=8.8 Hz, 1H), 4.33 (d, J=11.6 Hz, 1H), 4.12(dd, J=6.4, 11.7 Hz, 1H), 3.13-2.99 (m, 1H), 2.30 (dd, J=5.6, 16.1 Hz,1H), One non-exchangeable proton is obscured by the solvent peak and isnot tabulated. ¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ=−199.99 (s, 1F); ³¹PNMR (162 MHz, DEUTERIUM OXIDE) δ=55.96 (br. s., 1P), 51.76 (s, 1P)(internal standard H₃PO₄); LCMS [M+H]=678.

Peak 2: 30 mgs, 19% yield, 95% de, ¹H NMR (400 MHz, DEUTERIUM OXIDE)δ=8.45 (s, 1H), 8.27 (s, 1H), 8.21 (s, 1H), 7.68 (s, 1H), 6.49 (d,J=15.9 Hz, 1H), 6.33-6.14 (m, 1H), 5.55-5.43 (m, 1H), 5.13 (dd, J=10.9,14.9 Hz, 2H), 4.64 (d, J=5.3 Hz, 1H), 4.61 (br. s., 1H), 4.55 (d, J=8.9Hz, 1H), 4.36 (d, J=10.6 Hz, 1H), 4.16 (dd, J=5.9, 12.0 Hz, 1H),3.10-2.97 (m, 1H), 2.25 (dd, J=5.8, 15.8 Hz, 1H); ¹⁹F NMR (376 MHz,DEUTERIUM OXIDE) δ=−200.31 (s, 1F); ³¹P NMR (162 MHz, DEUTERIUM OXIDE)δ=55.98 (s, 1P), 50.38 (s, 1P) (H₃PO₄ internal standard); LCMS[M+H]=678.

Peak 3: 8 mgs, 7% yield, 93% de, ¹H NMR (400 MHz, DEUTERIUM OXIDE)δ=8.30 (s, 1H), 8.27 (s, 1H), 8.20 (s, 1H), 7.64 (s, 1H), 6.47 (d,J=16.5 Hz, 1H), 6.33-6.15 (m, 1H), 5.60-5.50 (m, 1H), 5.18-5.06 (m, 2H),4.63 (d, J=2.6 Hz, 2H), 4.55 (d, J=9.7 Hz, 1H), 4.43 (d, J=12.3 Hz, 1H),4.13-4.03 (m, 1H), 3.07-2.94 (m, 1H), 2.19 (dd, J=5.5, 15.8 Hz, 1H); ¹⁹FNMR (376 MHz, DEUTERIUM OXIDE) δ=−200.30 (s, 1F); ³¹P NMR (162 MHz,DEUTERIUM OXIDE) δ=51.72 (br. s., 1P), 50.31 (s, 1P) (H₃PO₄ internalstandard); LCMS [M+H]=678. Peak 4: 8 mgs, 6% yield, 93% de, ¹H NMR (400MHz, DEUTERIUM OXIDE) δ=8.29 (br. s., 1H), 8.27 (br. s., 1H), 8.19 (s,1H), 7.65 (br. s., 1H), 6.47 (d, J=16.0 Hz, 1H), 5.75-5.46 (m, 2H), 5.13(br. s., 2H), 4.69 (br. s., 1H), 4.62 (br. s., 1H), 4.53 (d, J=7.8 Hz,1H), 4.40 (d, J=10.8 Hz, 1H), 4.11-4.02 (m, 1H), 3.04 (br. s., 1H), 2.23(d, J=15.4 Hz, 1H); ¹⁹F NMR (376 MHz, DEUTERIUM OXIDE) δ=−199.96 (br.s., 1F); ³¹P NMR (162 MHz, DEUTERIUM OXIDE) δ=53.04 (br. s., 1P)(onlyone peak observed, H3PO4 internal standard); LCMS [M+H]=678.

Example 4(4S,6R,7S,11aR,13R,14R,14aR,15R)-6-(4-amino-7-methyl-1H-imidazo[4,5-c]pyridin-1-yl)-13-(6-amino-9H-purin-9-yl)-14-fluoro-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-15-ol2,9-dioxide

Example 4 is made in a similar fashion as Example 1 usingN-(7-methyl-1H-imidazo[4,5-c]pyridin-4-yl)benzamide in place of A-2 instep 1 of Scheme A.

Example 59-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-3-methyl-3,9-dihydro-6H-purin-6-one

Example 5 is made in a similar fashion as Example 1 using3-methyl-3,9-dihydro-6H-purin-6-one in place of A-2 in step 1 of SchemeA.

Example 63-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-4-methyl-3,4-dihydro-7H-imidazo[4,5-b]pyridin-7-one

Example 6 is made in a similar fashion as Example 1 using4-methyl-3,4-dihydro-7H-imidazo[4,5-b]pyridin-7-one in place of A-2 instep 1 of Scheme A.

Example 79-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-2-methyl-1,9-dihydro-6H-purin-6-one

Example 7 was made in a similar fashion as Example 3 using6-(benzyloxy)-2-methyl-9H-purine in place of F-1 in step 1 of Scheme F.

Peak 1—¹H NMR (400 MHz, Methanol-d₄) δ 8.72 (s, 1H), 8.26 (s, 1H), 8.25(s, 2H), 6.42 (d, J=15.8 Hz, 1H), 5.62 (dd, J=50.8, 3.7 Hz, 1H), 5.46(ddd, J=13.1, 9.1, 2.8 Hz, 1H), 5.28-5.14 (m, 2H), 4.80 (t, J=6.2 Hz,1H), 4.68 (s, 1H), 4.46 (d, J=9.0 Hz, 1H), 4.43-4.35 (m, 1H), 4.18 (dd,J=11.8, 5.7 Hz, 1H), 3.09-2.96 (m, 1H), 2.25 (s, 3H), 2.11 (dd, J=15.1,6.0 Hz, 1H); ¹⁹F NMR (376 MHz, MeOD) δ −200.3; ³¹P NMR (162 MHz, MeOD) δ59.20, 55.02; LCMS [M+H]=692.

Peak 2—¹H NMR (400 MHz, Methanol-d₄) δ 8.73 (s, 1H), 8.25 (s, 1H), 8.24(s, 1H), 6.41 (d, J=15.5 Hz, 1H), 6.22 (dd, J=50.3, 3.6 Hz, 1H),5.57-5.48 (m, 1H), 5.23-5.09 (m, 2H), 4.70 (s, 1H), 4.71-4.63 (m, 1H),4.45 (dd, J=16.5, 10.9 Hz, 2H), 4.23 (dd, J=11.7, 5.6 Hz, 1H), 2.95(ddd, J=11.8, 8.3, 4.7 Hz, 1H), 2.14 (dd, J=15.1, 6.0 Hz, 1H); ¹⁹F NMR(376 MHz, MeOD) δ −201.4; ³¹P NMR (162 MHz, MeOD) δ 59.33, 53.25; LCMS[M+H]=692.

Peak 3—¹H NMR (400 MHz, Methanol-d₄) δ 8.38 (s, 1H), 8.15 (s, 1H), 8.13(s, 1H), 6.29 (d, J=16.2 Hz, 1H), 6.12 (dd, J=50.3, 3.4 Hz, 1H), 5.36(t, J=9.4 Hz, 1H), 5.13-4.88 (m, 2H), 4.59 (d, J=5.8 Hz, 2H), 4.34 (t,J=10.5 Hz, 2H), 4.18 (dd, J=12.4, 6.4 Hz, 1H), 2.93-2.77 (m, 1H), 2.13(s, 3H), 1.98 (d, J=15.0 Hz, 1H); ¹⁹F NMR (376 MHz, MeOD) δ −201.2; ³¹PNMR (162 MHz, MeOD) δ 54.06, 52.75; LCMS [M+H]=692.

Peak 4—¹H NMR (400 MHz, Methanol-d₄) δ 8.35 (s, 1H), 8.13 (s, 1H), 8.11(s, 1H), 6.28 (d, J=16.5 Hz, 1H), 5.49 (dd, J=51.1, 3.6 Hz, 1H), 5.25(td, J=9.2, 2.8 Hz, 1H), 5.11 (td, J=10.3, 9.8, 5.7 Hz, 1H), 4.96 (ddt,J=23.4, 9.3, 4.8 Hz, 1H), 4.70 (t, J=6.2 Hz, 1H), 4.58 (s, 1H),4.37-4.27 (m, 2H), 4.17 (dd, J=12.3, 6.8 Hz, 1H), 2.92 (dddd, J=14.8,10.4, 6.1, 3.3 Hz, 1H), 2.19 (s, 3H), 1.92-1.86 (m, 1H); ¹⁹F NMR (376MHz, MeOD) δ −200.8; ³¹P NMR (162 MHz, MeOD) δ 55.16, 53.92; LCMS[M+H]=692.

Example 82-amino-9-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1,9-dihydro-6H-purin-6-one

Example 8 is made in a similar fashion as Example 1 usingN-(6-(benzyloxy)-9H-purin-2-yl)benzamide in place of A-2 in step 1 ofScheme A.

Example 95-amino-3-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]imidazo[4,5-d][1,3]oxazin-7(3H)-one

Example 9 is made in a similar fashion as Example 1 usingN-(7-oxo-3,7-dihydroimidazo[4,5-d][1,3]oxazin-5-yl)benzamide in place ofA-2 in step 1 of Scheme A.

Example 103-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-3,5-dihydro-9H-imidazo[1,2-a]purin-9-one

Example 10 is made in a similar fashion as Example 1 usingN-(6-(benzyloxy)-9H-purin-2-yl)benzamide in place of A-2 in step 1 ofScheme A.

Example 11(4S,6R,7S,11aR,13R,14R,14aR,15R)-6-(4-amino-3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-13-(6-amino-9H-purin-9-yl)-14-fluoro-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-15-ol2,9-dioxide

Example 11 is made in a similar fashion as Example 1 usingN-(3-methoxy-1H-pyrazolo[3,4-d]pyrimidin-4-yl)benzamide in place of A-2in step 1 of Scheme A.

Example 124-amino-1-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dioxido-2,9-disulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one

Example 12 is made in a similar fashion as Example 11 using anadditional deprotection step after step 5 of Scheme C.

Example 13(4S,6R,7S,11aS,13R,14R,14aR,15R)-6,13-bis(6-amino-9H-purin-9-yl)-14-fluoro-2-sulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,9,7,2,8]trioxathiadiphosphacyclotridecine-9,15-diol2,9-dioxide

Example 13 was made in a similar fashion as Example 1 using(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(mercaptomethyl)tetrahydrofuran-3-ylhydrogen phosphonate (H-2, Scheme H) in place of B-4, tetrazole in placeof pyridinium triflate (pyTFA), and tBuOOH in place of DDT in step 1 ofScheme C.

Peak 1: 30 mg, 24%; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.33 (s, 1H) 8.23(s, 1H) 8.15 (s, 1H) 8.13 (s, 1H) 6.15-6.26 (m, 1H) 5.66-5.85 (m, 1H)5.08-5.24 (m, 2H) 4.98-5.07 (m, 1H) 4.77 (t, J=7.40 Hz, 1H) 4.35 (br.s., 2H) 3.42 (d, J=14.31 Hz, 1H) 3.16-3.20 (m, 1H) 2.84 (br. s., 1H)2.57 (q, J=7.25 Hz, 1H) 1.79 (br. s., 1H); ³¹P NMR (162 MHz, DMSO-d6,internal reference H₃PO₄) δ ppm 51.14 (s, 1P) 9.89 (s, 1P), ¹⁹F NMR (376MHz, DMSO-d6) δ ppm −194.39 (s, 1F).

Peak 2: 18 mg, 15%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.39 (s, 1H) 8.21(s, 1H) 8.16 (s, 1H) 8.13 (s, 1H) 6.05-6.26 (m, 2H) 5.12-5.27 (m, 2H)4.93-5.03 (m, 1H) 4.62 (t, J=6.30 Hz, 1H) 4.31-4.42 (m, 2H) 3.41 (d,J=15.04 Hz, 1H) 3.10 (t, J=11.68 Hz, 1H) 2.85 (br. s., 1H) 1.68 (d,J=5.99 Hz, 1H); ³¹P NMR (162 MHz, DMSO-d6, internal reference H₃PO₄) δppm 48.59 (s, 1P) 10.51 (s, 1P), ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm−195.78 (s, 1F).

Step 1: Synthesis ofN-benzoyl-5′-S-benzoyl-2′-deoxy-2′-fluoro-5′-thioadenosine (H-1)

To a suspension of B-1 (2.00 g, 5.36 mmol) and thiobenzoic acid (1.11 g,8.04 mmol) in THF (50 mL) was added a solution of DIAD (1.48 mL, 7.50mmol) and PPh₃ (1.97 g, 7.50 mmol) in THF (5 mL). After stirringovernight, the reaction was diluted with EtOAc and washed with water andbrine. The organics were dried over MgSO₄, filtered and concentrated.The crude residue was purified via flash chromatography (80 g SiO₂,Isco, 0-100% EtOAc/heptanes) to afford H-1 (2.1 g, 79%). LCMS [M+H]=494observed; ¹H NMR (400 MHz, DMSO-d₆) δ ppm=11.21 (br. s., 1H) 8.74 (s,1H) 8.65 (s, 1H) 7.98-8.10 (m, 2H) 7.83-7.93 (m, 2H) 7.62-7.72 (m, 2H)7.50-7.61 (m, 4H) 6.39 (dd, J=19.20, 2.20 Hz, 1H) 5.97 (d, J=6.24 Hz,1H) 5.61-5.79 (m, 1H) 4.61-4.76 (m, 1H) 4.11-4.22 (m, 1H) 3.67 (dd,J=14.06, 4.28 Hz, 1H) 3.44 (dd, J=13.94, 7.21 Hz, 1H)

Step 2: Synthesis of(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(mercaptomethyl)tetrahydrofuran-3-ylhydrogen phosphonate (H-2)

H-1 (1.00 g, 2.03 mmol) was co-evaporated with anhydrous pyridine (3×)then the residue was dissolved a final time in anhydrous pyridine (20.0mL). The solution was cooled in an ice-water bath followed by theaddition of diphenyl phosphonate (770 uL, 4.05 mmol). The ice-bath wasremoved and the reaction was stirred for 2.5 hours. Another 1 eqdiphenyl phosphonate was added and after 1 hour, the reaction wasquenched with 1 M TEAB (gas evolved). Extracted with DCM (4×), driedorganics over dried over MgSO₄, filtered and concentrated. The residuewas dissolved in 33% MeNH₂ in EtOH (10 mL). After 30 min, the reactionwas concentrated and the crude residue was purified via flashchromatography (40 g SiO₂, Isco, 0-100% MeOH/DCM) to afford H-2 (2.1 g,79%). LCMS [M+H]=350 observed; ¹H NMR (400 MHz, DMSO-d₆) δ ppm=8.35 (s,1H), 8.16 (s, 1H), 7.36 (s, 2H), 6.24 (dd, J=2.6, 18.2 Hz, 1H),5.68-5.49 (m, 1H), 4.95-4.81 (m, 1H), 4.16-4.09 (m, 1H), 3.01-2.93 (m,1H), 2.86 (dd, J=6.1, 14.1 Hz, 1H); ³¹P NMR (162 MHz, DMSO-d₆) δppm=0.28 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=−201.15 (s, 1F).

Example 149-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-2,9,15-trihydroxy-2,9-dioxidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one

Example 14 was made in a similar fashion as Example 2 using ETT in placeof pyTFA, DCM in place of THF, and ^(t)BuOOH in place of DDTT in step 1and iodine in place of 3H-benzodithiol-3-one in step 2 of Scheme E. Thecrude material was purified by reverse phase chromatography (PhenomenexLuna Omega 5 um Polar column, Mobile phase A: H₂O w/10 mM NH₄OAc Mobilephase B: MeCN) to give the desired product. (11 mg, 13.8% yield)

¹H NMR (400 MHz, DMSO-d6) δ ppm 8.32 (s, 1H), 8.21 (s, 1H), 8.16 (s,1H), 8.04 (s, 1H), 6.27 (d, J=18.5 Hz, 1H), 5.59-5.39 (m, 1H), 5.15-5.07(m, 1H), 4.96-4.80 (m, 2H), 4.51-4.48 (m, 1H), 4.31-4.25 (m, 1H),4.23-4.17 (m, 1H), 4.11-4.05 (m, 2H), 3.46 (s, 3H), 2.90-2.76 (m, 1H),1.75-1.67 (m, 1H); ³¹P NMR (162 MHz, DMSO-d6, internal reference H₃PO₄)δ ppm −3.42 (s, 1P) −6.58 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm−198.30 (s, 1F)

Example 159-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-2,15-dihydroxy-2,9-dioxido-9-sulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one

Example 15 was made in a similar fashion as Example 2 using ETT in placeof pyTFA and DCM in place of THF in step 1 and iodine in place of3H-benzodithiol-3-one in step 2 of Scheme E. The crude material waspurified by reverse phase chromatography (Phenomenex Gemini NX-C18 3 umcolumn, Mobile phase A: H₂O w/10 mM NH₄OAc Mobile phase B: MeCN) to givethe two diastereomer products.

Peak 1

15 mg, 20% yield

¹H NMR (400 MHz, D₂O) δ ppm 8.49 (s, 1H), 8.33 (s, 1H), 8.21 (s, 1H),7.80 (s, 1H), 6.55 (d, J=16.0 Hz, 1H), 5.78-5.66 (m, 1H), 5.66-5.60 (m,2H), 5.18-5.11 (m, 2H), 4.70-4.59 (m, 1H), 4.59-4.54 (m, 1H), 4.43-4.37(m, 1H), 4.25-4.17 (m, 1H), 3.56 (s, 3H), 3.13-3.03 (m, 1H), 2.31-2.23(m, 1H); ³¹P NMR (162 MHz, DMSO-d6, internal reference H₃PO₄) δ ppm53.77 (s, 1P) −6.21 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −198.24(s, 1F)

Peak 2

27 mg, 29% yield

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.33 (s, 1H), 8.18 (s, 1H), 8.18 (s,1H), 8.02 (s, 1H), 6.28 (d, J=18.1 Hz, 1H), 5.55-5.38 (m, 1H), 5.35-5.24(m, 1H), 4.95-4.86 (m, 2H), 4.86-4.79 (m, 1H), 4.51-4.47 (m, 1H),4.29-4.22 (m, 2H), 4.12-4.08 (m, 1H), 3.46 (s, 3H), 2.87-2.76 (m, 1H),1.75-1.71 (m, 1H); ³¹P NMR (162 MHz, DMSO-d6, internal reference H₃PO₄)δ ppm 49.60 (s, 1P) −6.27 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm−199.37 (s, 1F)

Example 169-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-9,15-dihydroxy-2,9-dioxido-2-sulfanyloctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one

Example 16 was made in a similar fashion as Example 2 using ETT in placeof pyTFA, DCM in place of THF, and ^(t)BuOOH in place of DDTT in step 1of Scheme E. The crude material was purified by reverse phasechromatography (Phenomenex Gemini NX-C18 3 um column, Mobile phase A:H₂O w/10 mM NH₄OAc Mobile phase B: MeCN) to give the two diastereomerproducts.

Peak 1

11 mg, 8.5% yield

¹H NMR (400 MHz, D₂O) δ ppm 8.36 (s, 1H), 8.33 (s, 1H), 8.19 (s, 1H),7.71 (s, 1H), 6.53 (d, J=16.4 Hz, 1H), 5.80-5.60 (m, 3H), 5.25-5.11 (m,2H), 4.68 (m, 1H), 4.59-4.53 (m, 1H), 4.43 (m, 1H), 4.20-4.13 (m, 1H),3.55 (s, 3H), 3.14-3.02 (m, 1H), 2.26-2.18 (m, 1H); ³¹P NMR (162 MHz,DMSO-d6, internal reference H₃PO₄) δ ppm 50.40 (s, 1P) −3.59 (s, 1P);¹⁹F NMR (376 MHz, DMSO-d6) ppm −197.41 (s, 1F)

Peak 2

32 mg, 24.3% yield

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.35 (s, 1H), 8.28 (s, 1H), 8.15 (s,1H), 8.13 (s, 1H), 6.27 (d, J=17.4 Hz, 1H), 6.20-6.01 (m, 1H), 5.07-4.99(m, 1H), 4.98-4.83 (m, 2H), 4.48-4.37 (m, 2H), 4.26-4.19 (m, 1H),4.17-4.09 (m, 1H), 4.08-3.99 (m, 1H), 3.47 (s, 3H), 2.90-2.79 (m, 1H),1.68-1.60 (m, 1H); ³¹P NMR (162 MHz, DMSO-d₆, internal reference H₃PO₄)δ 48.53 (s, 1P) −3.30 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d₆) −198.19 (s,1F)

Example 179-[(4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-9-oxido-2,9-disulfanyl-2-sulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9,2,8]tetraoxadiphosphacyclotridecin-6-yl]-1-methyl-1,9-dihydro-6H-purin-6-one

Example 17 was made in a similar fashion as Example 1 using compound I-1(N-benzoyl-2′-deoxy-2′-fluoro-3′-O-[hydroxy(sulfido)-I⁵-phosphanyl]adenosine)in place of B-4, ETT in place of pyTFA, DCM in place of THF in step 1and DPPCI in place of DMOCP in step 2 of Scheme C.

Purification: Phenomenex Gemini NX-C18 3 um column, Mobile phase A: H₂Ow/10 mM NH₄OAc Mobile phase B: MeCN

Peak 1

7.4 mg, 7.7% yield

¹H NMR (400 MHz, D₂O) δ ppm 8.51 (s, 1H), 8.33 (s, 1H), 8.21 (s, 1H),7.74 (s, 1H), 6.56 (d, J=15.6 Hz, 1H), 6.47-6.28 (m, 1H), 5.80-5.69 (m,1H), 5.40-5.26 (m, 1H), 5.22-5.10 (m, 2H), 4.65-4.62 (m, 1H), 4.62-4.56(m, 1H), 4.46-4.39 (m, 1H), 4.24-4.16 (m, 1H), 3.52 (s, 3H), 3.17-3.03(m, 1H), 2.33-2.22 (m, 1H); ³¹P NMR (162 MHz, D₂O, internal referenceH₃PO₄) δ 108.39 (s, 1P) 56.07 (s, 1P); ¹⁹F NMR (376 MHz, D₂O) −199.57(s, 1F)

Peak 2

9.4 mg, 8.5% yield

¹H NMR (400 MHz, D₂O) δ ppm 8.36 (s, 1H), 8.32 (s, 1H), 8.20 (s, 1H),7.69 (s, 1H), 6.54 (d, J=16.0 Hz, 1H), 6.46-6.30 (m, 1H), 5.86-5.75 (m,1H), 5.34-5.20 (m, 2H), 5.16-5.10 (m, 1H), 4.68-4.64 (m, 1H), 4.63-4.58(m, 1H), 4.53-4.45 (m, 1H), 4.17-4.08 (m, 1H), 3.52 (s, 3H), 3.16-2.99(m, 1H), 2.27-2.17 (m, 1H); ³¹P NMR (162 MHz, D₂O, internal referenceH₃PO₄) δ 108.06 (s, 1P) 51.78 (s, 1P); ¹⁹F NMR (376 MHz, D₂O) −199.73(s, 1F)

Compound I-1N-benzoyl-2′-deoxy-2′-fluoro-3′-O-[hydroxy(sulfido)-I⁵-phosphanyl]adenosine

Compound I-1 was made in a similar fashion as example B-4 according toliterature proceedures (Jones et al. Nucleosides, Nucleotides andNucleic Acids 2009, 28, 352-378) using diphenyl phosphonate in place ofditbutyl phosphonate and Li₂S in place of water in step 2 of Scheme B.

Example 189-((4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dimercapto-2,9-disulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecin-6-yl)-1-methyl-1,9-dihydro-6H-purin-6-one(J-5)

Step 1: Synthesis ofO-[(1S,2R,3S,5R)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl]S-[(2,4-dichlorophenyl)methyl] N,N-dimethylphosphoramidothioite (J-1)

All solutions contained powdered molecular sieves. To a cooled(ice-water bath) solution of N,N-dimethylphosphoramidous dichloride(0.34 mL, 2.9 mmol) in DCM (10 mL) was added a solution of A-8 (1.0 g,1.5 mmol) and DIEA (2.0 mL, 12 mmol) in DCM (10 mL). After 1 hr, asolution of (2,4-dichlorophenyl)methanethiol (0.83 mL, 5.9 mmol) in DCM(5 mL) was added and the ice-bath was removed. After 1 hr, the molecularsieves were removed by filtration and the filtrate was concentrated thenpurified via flash chromatography (40 g SiO₂, Isco, 0-100%EtOAc/heptanes) to afford J-1 (790 mg, 57%) as a white solid. ³¹P NMR(162 MHz, DMSO-d₆) δ ppm=174.1 2, 169.6

Step 2: Synthesis ofN-benzoyl-5′-O-([({[(1S,2R,3S,5R)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-2-{[tert-butyl(dimethyl)silyl]oxy}-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl]oxy}{[(2,4-dichlorophenyl)methyl]sulfanyl}phosphorothioyl)oxy]{[(2,4-dichlorophenyl)methyl]sulfanyl}phosphorothioyl)-2′-deoxy-2′-fluoroadenosine(J-2)

A mixture of J-1 (1.3 g, 1.3 mmol) and powdered molecular sieves in DCM(13 mL) was stirred for 20 min (flask A). In a separated flask a mixtureof B-1 (540 mg, 1.5 mmol), ETT (1.3 g, 9.9 mmol) and powdered molecularsieves in DMF (13 mL) was stirred for 20 min (flask B). The contents offlask B were then added to flask A. After 30 min, DDTT (310 mg, 1.5mmol) was added. After 15 min, the molecular sieves were removed byfiltration and the filtrate was concentrated then purified via flashchromatography (40 g SiO₂, Isco, 0-100% EtOAc/heptanes) to afford J-2(436 mg, not pure). LCMS [M+H]=1308 observed; ³¹P NMR (162 MHz, DMSO-d₆)δ ppm=95.8, 94.3; ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=201.49, 201.51

Step 3: Synthesis ofO-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((1S,2R,3S,5R)-2-((tert-butyldimethylsilyl)oxy)-3-hydroxy-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)cyclopentyl)oxy)((2,4-dichlorobenzyl)thio)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl)S-hydrogen phosphonothioate (J-3)

A mixture of J-2 (180 mg, 0.14 mmol, not pure) sulfur (13 mg, 0.42 mmol)and N,N-diethylethanaminium phosphinate (0.120 mL, 0.83 mmol) wasco-evaporated with pyridine. The residue was dissolved in pyridine (1.4mL) then DMOCP (77 mg, 0.42 mmol) was added.

After ˜1 hr, the reaction was diluted with EtOAc, washed with water andbrine and concentrated. To the residue was added DCM (2 mL) followed bydichloroacetic acid (120 uL) resulting in a bright orange solution.After 15 min, quenched with pyridine until orange color dissipated,concentrated and purified via flash chromatography (12 g SiO₂, Isco,0-30% MeOH/DCM) to afford J-3 (64 mg). LCMS [M+H]=1086 observed; ³¹P NMR(162 MHz, DMSO-d₆) δ ppm=96.9, 94.2, 49.4; 48.7; ¹⁹F NMR (376 MHz,DMSO-d₆) δ ppm=199.6, 199.7, 199.8, 200.1

Step 4: Synthesis ofN-(9-((4S,6R,7S,11aR,13R,14R,14aR,15R)-15-((tert-butyldimethylsilyl)oxy)-9-((2,4-dichlorobenzyl)thio)-14-fluoro-2-mercapto-6-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,9-disulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecin-13-yl)-9H-purin-6-yl)benzamide(J-4)

J-4 was made in a similar fashion as C-2 using DPPCl in place of DMOCP.LCMS [M+H]=1100 observed; ³¹P NMR (162 MHz, DMSO-d₆) δ ppm=110.4, 110.3,97.1, 95.5; ¹⁹F NMR (376 MHz, DMSO-d₆) δ ppm=196.0, 196.5

Step 5: Synthesis of9-((4S,6R,7S,11aR,13R,14R,14aR,15R)-13-(6-amino-9H-purin-9-yl)-14-fluoro-15-hydroxy-2,9-dimercapto-2,9-disulfidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecin-6-yl)-1-methyl-1,9-dihydro-6H-purin-6-one(J-5)

To a flask containing J-4 (24 mg, 0.022 mmol) was added a 1:1 solution(0.5 mL) of ACN and 33% methyl amine in EtOH. After 3 hrs, the reactionwas concentrated and the residue was dissolved in a 1:1:2 solution (0.2mL) of thiophenol, TEA, and dioxane. After 5 hrs, the reaction wasconcentrated. To the residue was added a 1:1 solution (0.4 mL) ofTEA:pyridine followed by triethylamine trihydrofluoride (150 uL). Thereaction was heated to 70° C. for 12 hrs then quenched with saturatedsolution of sodium bicarbonate and concentrated. The residue wastriturated with 10% MeOH/DCM then Et₂O (2×) then purified by reversephase prep-HPLC [Phenomenex Gemini NX-C18 5 um 21×150 mm column elutingwith 0-80% MeCN/H₂O containing NH₄HCO₃ (10 mM)]

to give 5 mg of J-5.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.54 (s, 1H) 8.19 (s, 1H) 8.17 (s, 1H)8.04 (s, 1H) 6.30 (d, J=16.02 Hz, 1H) 6.03-6.20 (m, 1H) 5.24-5.35 (m,1H) 4.94-5.10 (m, 2H) 4.75-4.81 (m, 1H) 4.36-4.40 (m, 1H) 4.29-4.35 (m,1H) 4.17-4.25 (m, 1H) 3.88-3.95 (m, 1H) 3.49 (s, 3H) 2.80-2.91 (m, 1H)1.61-1.68 (m, 1H); ³¹P NMR (162 MHz, DMSO-d₆) δ ppm 113.20, 110.74; ¹⁹FNMR (376 MHz, DMSO-d₆) δ ppm −198.74 LCMS [M+H]=724 observed

Example 199,9′-((4S,6R,7S,11aR,13R,14R,14aR,15R)-14-fluoro-15-hydroxy-2,9-dimercapto-2,9-dioxidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecine-6,13-diyl)bis(1-methyl-1,9-dihydro-6H-purin-6-one

Example 19 was made in a similar fashion as Example 17 using(2R,3R,4R,5R)-4-fluoro-2-(hydroxymethyl)-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate K-4 in place of B-4. The crude material waspurified by reverse phase chromatography (Phenomenex Luna Omega 5 umPolar C18 4.6×50 mm column; Mobile phase A: H2O w/10 mM NH4OAc, Mobilephase B: MeCN; elution with a gradient of 0-10% B in 2.0 minutes, thenramp 10-80% at 5.5 min, hold 80% for 0.5 minutes then re-equilibrate;Flow 2.25 mL/min) to give the four diastereomer products.

Peak 1:38 mg, 10.6%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.45 (s, 1H) 8.26(s, 1H) 8.18 (s, 1H) 8.16 (s, 1H) 6.27 (d, J=18.58 Hz, 1H) 5.51-5.68 (m,1H) 5.48 (d, J=2.57 Hz, 1H) 5.01-5.10 (m, 1H) 4.95 (td, J=9.75, 5.69 Hz,2H) 4.60 (br. s., 1H) 4.55 (t, J=5.62 Hz, 1H) 4.20 (br. s., 1H)3.95-4.02 (m, 1H) 3.86-3.94 (m, 1H) 3.52 (s, 3H) 3.51 (s, 3H) 2.82 (d,J=11.62 Hz, 1H) 1.69-1.75 (m, 1H); ³¹P NMR (162 MHz, DMSO-d6) δ ppm53.70 (s, 1P) 49.98 (s, 1P); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −196.72(s., 1F); LCMS [M+H]=707.0.

Peak 2: 35 mg, 9.3%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.44 (s, 1H) 8.27(s, 1H) 8.22 (s, 1H) 8.21 (s, 1H) 6.10-6.26 (m, 2H) 4.89-5.06 (m, 3H)4.61 (br. s., 1H) 4.44 (t, J=5.32 Hz, 1H) 4.20 (d, J=8.80 Hz, 1H) 3.97(d, J=2.32 Hz, 2H) 3.52 (s, 3H) 3.51 (s, 3H) 2.75-2.84 (m, 1H) 1.67 (dd,J=14.73, 5.07 Hz, 1H); ³¹P NMR (162 MHz, DMSO-d6) δ ppm 53.60 (br. s.,1P) 48.19 (br. s., 1P); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −197.17 (s.,1F); LCMS [M+H]=707.0.

Peak 3: 8 mg, 2%; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.47 (s, 1H) 8.24 (s,1H) 8.21 (s, 1H) 8.10 (s, 1H) 6.52 (s, 1H) 6.26 (d, J=17.61 Hz, 1H)5.45-5.65 (m, 1H) 5.12-5.25 (m, 2H) 4.81-4.97 (m, 2H) 4.51 (t, J=6.30Hz, 1H) 4.45 (br. s., 1H) 4.23 (d, J=8.56 Hz, 1H) 3.98-4.13 (m, 2H) 3.53(s, 3H) 3.50 (s, 3H) 2.74-2.87 (m, 1H) 1.67 (dd, J=14.67, 6.11 Hz, 1H);³¹P NMR (162 MHz, DMSO-d6) δ ppm 50.04 (s, 1P) 48.32 (br. s., 1P); ¹⁹FNMR (376 MHz, DMSO-d6) δ ppm −198.02 (s., 1F); LCMS [M+H]=707.0.

Peak 4: 40 mg, 10.9%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.46 (s, 1H) 8.32(s, 1H) 8.24 (s, 1H) 8.16 (s, 1H) 6.27 (d, J=17.48 Hz, 1H) 6.07-6.23 (m,1H) 5.19 (td, J=9.51, 2.63 Hz, 1H) 4.81-4.96 (m, 3H) 4.45 (br. s., 1H)4.40 (t, J=5.62 Hz, 1H) 4.24 (d, J=9.41 Hz, 1H) 4.12 (d, J=11.98 Hz, 1H)4.01 (dd, J=11.68, 5.32 Hz, 1H) 3.53 (s, 3H) 3.50 (s, 3H) 2.81 (d,J=7.46 Hz, 1H) 1.61 (dd, J=14.55, 6.24 Hz, 2H); ³¹P NMR (162 MHz,DMSO-d6) δ ppm 49.20 (br. s., 1P) 48.19 (br. s., 1P); ¹⁹F NMR (376 MHz,DMSO-d6) δ ppm −198.36 (s., 1F); LCMS [M+H]=707.0.

Step 1: Synthesis of 2′-deoxy-2′-fluoro-1-methylinosine (K-2)

Compound K-2 was made in a similar fashion as D-4 using2′-deoxy-2′-fluoroinosine (K-1) in place of D-3 in step 3 of Scheme D in98% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.44 (s, 1H) 8.35 (s, 1H) 6.21 (dd,J=16.75, 2.69 Hz, 1H) 5.73 (d, J=6.11 Hz, 1H) 5.25-5.47 (m, 1H) 5.14 (t,J=5.44 Hz, 1H) 4.36-4.52 (m, 1H) 3.93-4.04 (m, 1H) 3.75 (ddd, J=12.32,5.17, 2.81 Hz, 1H) 3.59 (ddd, J=12.32, 5.65, 4.03 Hz, 1H) 3.52 (s, 3H);19F NMR (376 MHz, DMSO-d6) δ ppm −204.63 (s, 1F); LCMS [M+H]=285.1.

Step 2: Synthesis of5′-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2′-deoxy-2′-fluoro-1-methylinosine(K-3)

Compound K-3 was made in a similar fashion as B-2 using microwave at100° C. for 10 min instead of 25° C. for 12 h in step 1 of Scheme B in60% yield.

¹H NMR (400 MHz, DMSO-d6) δ ppm 8.39 (s, 1H) 8.24 (s, 1H) 7.29-7.36 (m,2H) 7.15-7.28 (m, 7H) 6.81 (dd, J=8.86, 7.64 Hz, 4H) 6.27 (dd, J=19.44,1.47 Hz, 1H) 5.72 (d, J=6.85 Hz, 1H) 5.38-5.59 (m, 1H) 4.55-4.72 (m, 1H)4.10 (dt, J=7.76, 3.94 Hz, 1H) 3.72 (d, J=1.71 Hz, 6H) 3.51 (s, 3H)3.20-3.28 (m, 2H); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −199.30 (s, 1F);LCMS [M+H]=587.2.

Step 3: Synthesis of(2R,3R,4R,5R)-4-fluoro-2-(hydroxymethyl)-5-(1-methyl-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-ylhydrogen phosphonate (K-4)

K-3 (1.28 g, 2.19 mmol) was co-evaporated with anhydrous pyridine (3×)then the residue was dissolved a final time in anhydrous pyridine (22.0mL). The solution was added dropwise to a solution of diphenylphosphonate (3.6 g, 15.4 mmol) in anhydrous pyridine (22.0 mL) in anoven dried flask. The reaction was stirred at rt for 15 min under N₂,triethylamine-water (12 mL, 1:1, v/v) was added, continued to stir at rtfor 15 min, The reaction mixture was concentrated by vacuum, dissolvedin DCM (22.0 mL), DCA (5.66 g, 43.9 mmol) was added, stirred at rt for15 min, then quenched by pyridine (22.0 mL). The reaction wasconcentrated, purified via flash chromatography (40 g SiO₂, Isco, 50%MeOH/DCM) to afford K-4 (0.71 g, 93%) as 0.8 eq. Et₃N salt.

¹H NMR (400 MHz, DMSO-d6) δ ppm 8.60 (br. s., 1H) 8.44 (s, 1H) 8.34 (s,1H) 7.84 (t, J=7.58 Hz, 0.36H) 7.61 (s, 0.5H) 7.38-7.49 (m, 0.75H) 6.25(d, J=15.89 Hz, 1H) 6.01 (s, 0.5H) 5.75 (s, 0.2H) 5.43-5.66 (m, 1H)4.92-5.09 (m, 1H) 4.14 (br. s., 1H) 3.75 (d, J=11.13 Hz, 1H) 3.64 (d,J=12.72 Hz, 1H) 3.51 (br. s., 3H) 3.04-3.13 (m, 1.7H) 1.17 (t, J=7.15Hz, 2.5H); ³¹P NMR (162 MHz, DMSO-d6) δ ppm 1.88 (br. s., 1P); ¹⁹F NMR(376 MHz, DMSO-d6) δ ppm −200.26 (br. s., 1F). LCMS [M+H]=350 observed;LCMS [M+H]=349.0.

Example 209,9′-((4S,6R,7S,11aR,13R,14R,14aR,15R)-14-fluoro-2,15-dihydroxy-9-mercapto-2,9-dioxidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecine-6,13-diyl)bis(1-methyl-1,9-dihydro-6H-purin-6-one)

Example 20 was made in a similar fashion as Example 15. The crudematerial was purified by reverse phase chromatography (Phenomenex GeminiNX-C18 4.6×50 mm 5 um column; Mobile phase A: H2O w/10 mM NH4OAc, Mobilephase B: MeCN; elution with a gradient of 0-80% B in 5.0 minutes, hold80% for 0.5 minutes then re-equilibrate; Flow 2.25 mL/min) to give thetwo diastereomer products.

Peak 1:33 mg, 23%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.44 (s, 1H) 8.27 (s,1H) 8.21 (s, 1H) 8.15 (s, 1H) 6.25 (d, J=18.58 Hz, 1H) 5.43-5.67 (m, 2H)4.99-5.07 (m, 1H) 4.89-4.98 (m, 1H) 4.76-4.88 (m, 1H) 4.68 (br. s., 1H)4.26 (br. s., 1H) 4.17 (br. s., 1H) 3.95-4.03 (m, 1H) 3.87-3.95 (m, 1H)3.52 (s, 3H) 3.52 (s, 3H) 2.76 (br. s., 1H) 1.67 (dd, J=14.79, 5.38 Hz,1H); ³¹P NMR (162 MHz, DMSO-d6) δ ppm 53.57 (s, 1P) −5.78 (br. s., 1P);¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −197.49 (br. s., 1F); LCMS [M+H]=691.0.

Peak 2: 33 mg, 23%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.47 (s, 1H) 8.25(s, 1H) 8.24 (s, 1H) 8.15 (s, 1H) 7.11 (br. s., 2H) 6.52 (s, 3H) 6.25(d, J=17.36 Hz, 1H) 5.42-5.62 (m, 1H) 5.19 (d, J=3.42 Hz, 2H) 4.88 (td,J=9.96, 6.36 Hz, 1H) 4.68-4.81 (m, 1H) 4.52 (br. s., 1H) 4.17-4.27 (m,2H) 3.97-4.14 (m, 2H) 3.53 (s, 3H) 3.51 (s, 3H) 2.72-2.84 (m, 1H) 1.61(dd, J=14.49, 6.05 Hz, 1H); ³¹P NMR (162 MHz, DMSO-d6) δ ppm 48.70 (br.s., 1P) −5.56 (br. s., 1P); ¹⁹F NMR (376 MHz, DMSO-d6) δ ppm −196.64(br. s., 1F); LCMS [M+H]=691.0.

Example 219,9′-((4S,6R,7S,11aR,13R,14R,14aR,15R)-14-fluoro-9,15-dihydroxy-2-mercapto-2,9-dioxidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecine-6,13-diyl)bis(1-methyl-1,9-dihydro-6H-purin-6-one

Example 21 was made in a similar fashion as Example 16. The crudematerial was purified by reverse phase chromatography (Phenomenex GeminiNX-C18 3 um 4.6×50 mm column; Mobile phase A: H2O w/10 mM NH₄OAc, Mobilephase B: MeCN; elution with a gradient of 0-80% B in 5.0 minutes, hold80% for 0.5 minutes then re-equilibrate; Flow 2.25 mL/min) to give thetwo diastereomer products.

Peak 1: 7.8 mg, 20%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.38 (s, 1H) 8.31(s, 1H) 8.21 (s, 2H) 6.10-6.30 (m, 2H) 5.83 (br. s., 1H) 4.88 (br. s.,3H) 4.47 (d, J=9.54 Hz, 2H) 4.16 (d, J=9.17 Hz, 1H) 3.96-4.11 (m, 2H)3.52 (s., 3H) 3.51 (s., 3H) 2.80 (m, 1H) 1.67 (m, 1H); ³¹P NMR (162 MHz,DMSO-d6) δ ppm 47.81 (s, 1P) −3.08 (br. s., 1P); ¹⁹F NMR (376 MHz,DMSO-d6) δ ppm −200.26 (br. s., 1F); LCMS [M+H]=691.0.

Peak 2: 4 mg; 10%; ¹H NMR (400 MHz, DMSO-d6) δ ppm 8.38 (s, 1H) 8.19 (s,2H) 8.15 (s, 1H) 6.24 (d, J=18.22 Hz, 1H) 5.78 (d, J=2.69 Hz, 1H)5.49-5.65 (m, 1H) 4.80-4.92 (m, 3H) 4.55 (s, 1H) 4.48 (br. s., 1H) 4.15(d, J=8.44 Hz, 1H) 4.08 (d, J=12.35 Hz, 1H) 3.97 (d, J=8.68 Hz, 1H) 3.52(s, 3H) 3.51 (s, 3H) 2.78 (m, 1H) 1.72 (d, J=11.13 Hz, 1H); ³¹P NMR (162MHz, DMSO-d6) δ ppm 49.78 (s, 1P) −3.06 (s, 1P); ¹⁹F NMR (376 MHz,DMSO-d6) δ ppm −195.17 (s, 1F); LCMS [M+H]=691.0.

Example 229,9′-((4S,6R,7S,11aR,13R,14R,14aR,15R)-14-fluoro-2,9,15-trihydroxy-2,9-dioxidooctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecine-6,13-diyl)bis(1-methyl-1,9-dihydro-6H-purin-6-one)

Example 22 was made in a similar fashion as Example 14 using theH-phosphonate K-4 in place of B-4. The crude material was purified byreverse phase chromatography (Phenomenex Gemini NX-C18 3 um 4.6×50 mmcolumn; Mobile phase A: H2O w/10 mM NH₄OAc, Mobile phase B: MeCN;elution with a gradient of 0-80% B in 5.0 minutes, hold 80% for 0.5minutes, then re-equilibrate; Flow 2.25 mL/min) to give the desiredproduct.

¹H NMR (400 MHz, DMSO-d6) δ ppm 8.40 (s, 1H) 8.21 (s, 1H) 8.21 (s, 1H)8.19 (s, 1H) 6.24 (d, J=17.97 Hz, 1H) 5.74 (br. s., 1H) 5.44-5.64 (m,1H) 4.83-4.96 (m, 2H) 4.66-4.81 (m, 1H) 4.57 (br. s., 1H) 4.27 (d,J=4.77 Hz, 1H) 4.06-4.18 (m, 2H) 3.96-4.04 (m, 1H) 3.52 (s, 3H) 3.52 (s,3H) 2.71-2.82 (m, 1H) 1.67 (dd, J=13.94, 5.26 Hz, 1H); ³¹P NMR (162 MHz,DMSO-d6) δ ppm −3.03 (s, 1P) −5.74 (br. s., 1P); ¹⁹F NMR (376 MHz,DMSO-d6) δ ppm −196.01 (br. s., 1F); LCMS [M+H]=675.0.

Example 23(4S,6R,7S,11aR,13R,14R,14aR,15R)-6,13-bis(6-amino-9H-purin-9-yl)-14-fluoro-2,9,15-trihydroxyoctahydro-11H-4,7-methanofuro[3,2-d][1,3,7,9]tetraoxa[2,8]diphosphacyclotridecine2,9-dioxide

Example 23 was made in a similar fashion as Example 14 using thephosphoramidite A-7 in place of D-8. The crude material was purified byreverse phase chromatography using an Agela Durashell C18 25×150 mmcolumn eluting with 0-10% MeCN/H₂O containing NH₄CO₃(10 mM). LCMS TOF(ESI+) [M+H]⁺=645 observed; 1H NMR (400 MHz, D₂O) δ ppm=8.31-8.24 (m,3H), 7.86 (s, 1H), 6.47 (d, J=16.8 Hz, 1H), 5.76-5.55 (m, 1H), 5.42-5.29(m, 1H), 5.22-5.12 (m, 1H), 5.10-4.97 (m, 1H), 4.68 (d, J=0.8 Hz, 1H),4.52 (d, J=7.0 Hz, 2H), 4.37 (d, J=12.3 Hz, 1H), 4.13 (dd, J=5.5, 12.3Hz, 1H), 3.12-2.98 (m, 1H), 2.21 (dd, J=4.9, 15.4 Hz, 1H); ³¹P NMR (162MHz, D₂O) δ ppm=−4.21 (s, 2P); ¹⁹F NMR (376 MHz, D₂O) δ ppm=−200.68 (brs, 1F).

Biological Examples Biochemical Assay Methods Surface Plasmon Resonance(SPR) Binding

Surface plasmon resonance (SPR) STING agonist binding studies werecarried out using a Biacore T200 instrument (GE Healthcare) at 4° C. ina 150 mM KCl, 25 mM Hepes (pH 7.5), 1 mM TCEP, 2.5 mM MgCl2, 5% (v/v)glycerol, 0.005% (v/v) P20, 1% (v/v) DMSO running buffer. Therecombinant protein immobilized on the streptavidin chip was eitherhuman WT or H232R STING. A truncated construct of STING was used in allstudies. The STING constructs were comprised of residues 155-341 withboth N- and C-terminal truncations; the N-terminal transmembrane domainswere removed (1-154), as well as the C-terminal tail (342-379). A highlyspecific N-terminal biotinylation was achieved enzymatically with the E.coli biotin ligase (BirA) and inclusion of the high-affinitybiotinylation peptide AviTag™. A Carboxymethylated dextranpre-immobilized with streptavidin (series S Streptavidin CM5 SensorChip) was used to capture the biotinylated STING protein. Test compoundinjections were made at a flow rate of 100 μl per minute with a 60second association time and variable dissociation time. A three-folddilution series from a 10 μM starting concentration was used for alltest compounds. Data analysis was performed using the BiacoreT200 dataevaluation software package (GE Healthcare). Compound injections werereferenced to both a blank surface and a buffer blank. Processed datawere fit to an equilibrium or kinetic model to obtain the observeddissociation constant K_(D). SPR binding data is provided in Table 1.

TABLE 1 H232R WT STING K_(D) STING K_(D) Mean (μM) Mean (μM) SPR binding(N) (N) Example 1 Peak 1   4.1 (2)   4.7 (2) Example 1 Peak 2  3.25 (2)  2.75 (2) Example 1 Peak 3   1.5 (3)   0.48 (3) Example 1 Peak 4  0.58(3)   0.24 (3) Example 2 Peak 1  0.116 (1)  0.023 (1) Example 2 Peak 2 0.091 (1)  0.093 (1) Example 2 Peak 3  .006 (1)  .00044 (1) Example 2Peak 4  0.011 (1) 0.00078 (1) Example 3 Peak 1  0.032 (1)  0.008 (1)Example 3 Peak 2  0.039 (1)  0.014 (1) Example 3 Peak 3  0.003 (1) 0.001 (1) Example 3 Peak 4  0.001 (1)  0.0003 (1) Example 7 Peak 1 1.04 (1)  0.098 (1) Example 7 Peak 2  1.89 (1)  0.416 (1) Example 7Peak 3  0.009 (1)  0.004 (1) Example 7 Peak 4  0.003 (1)  0.003 (1)Example 13 Peak 1  1.21 (2)   1.29 (3) Example 13 Peak 2  4.57 (2)  5.98 (3) Example 14  0.216 (2)  0.015 (2) Example 15 Peak 1  0.102 (2) 0.045 (2) Example 15 Peak 2  0.006 (2)  0.003 (2) Example 16 Peak 1 0.003 (2)  0.057 (2) Example 16 Peak 2  0.132 (2)  0.020 (2) Example 17Peak 1  0.004 (1)  0.0015 (2) Example 17 Peak 2 0.0005 (2)  0.0003 (2)Example 18  0.001 (1)  0.0002 (1) Example 19 Peak 1  0.102 (1)  0.086(1) Example 19 Peak 2  0.750 (1)  0.740 (1) Example 19 Peak 3  0.002 (1) 0.003 (1) Example 19 Peak 4  0.010 (1)  0.019 (1) Example 20 Peak 1 0.447 (1)  0.256 (1) Example 20 Peak 2  0.029 (2)  0.046 (2) Example 21Peak 1  0.198 (1)  0.127 (1) Example 21 Peak 2  0.048 (1)  0.016 (1)Example 22  0.117 (1)  0.449 (1) Example 23  5.05 (1)  0.993 (1)

Scintillation Proximity Assay (SPA) Competitive Binding

A radioligand binding assay was developed to determine compoundinteractions were competitive with a tritium-labeled version of thenative STING ligand, ³H-cyclic guanine (2′,5′) monophosphate adenine(3′,5′) monophosphate (³H-cGAMP). The STING constructs (WT and H232R)were comprised of residues 155-341 with both N- and C-terminaltruncations; the N-terminal transmembrane domains were removed (1-154),as well as the C-terminal tail (342-379). A highly specific N-terminalbiotinylation was achieved enzymatically with the E. coli biotin ligase(BirA) and inclusion of the high-affinity biotinylation peptide AviTag™.100 nM STING protein was immobilized on 20 μg streptavidin polyvinyltoluene (SA-PVT) beads in 150 mM NaCl, 25 mM Hepes (pH 7.5), 0.1 mMEDTA, 1 mM DTT, 0.005% (v/v) Tween-20, 1% (v/v) DMSO. 100 nM ³H-cGAMPand compounds were added and allowed to come to equilibrium at roomtemperature (20 min). Compounds were tested in three-fold dilutionseries from a 100 μM starting concentration and normalized to a positivecontrol compound that completely blocked ³H-cGAMP binding and thenegative control DMSO. The K_(I) for competitive binding was determinedfrom the IC₅₀ with the Cheng-Prusoff equation (Cheng & Prusoff,Biochemical Pharmacology, 22 (1973), pp. 3099-3108). The K_(D) valuesfor ³H-cGAMP used in the Cheng-Prusoff equation were determinedempirically to be 1 nM for WT STING, and 750 nM for R232H STING. SPAcompetitive binding data is provided in Table 2.

TABLE 2 WT STING K_(I) SPA competitive Mean (μM) binding (N) Example 1Peak 3 0.107 (8) Example 1 Peak 4 0.042 (8) Example 2 Peak 1 0.007 (5)Example 2 Peak 2 0.073 (5) Example 2 Peak 3 0.002 (9) Example 2 Peak 40.0002 (9) Example 3 Peak 1 0.004 (2) Example 3 Peak 2 0.018 (2) Example3 Peak 3 0.001 (1) Example 3 Peak 4 0.0002 (4) Example 7 Peak 1 0.044(1) Example 7 Peak 2 0.107 (1) Example 7 Peak 3 0.013 (1) Example 7 Peak4 0.003 (1) Example 13 Peak 1 >1 (2) Example 13 Peak 2 ( >1 (2) Example14 0.008 (3) Example 15 Peak 1 0.037 (3) Example 15 Peak 2 0.001 (3)Example 16 Peak 1 0.001 (3) Example 16 Peak 2 0.009 (3) Example 17 Peak1 0.002 (1) Example 17 Peak 2 0.0003 (2) Example 18 0.00019 (1) Example19 Peak 1 0.022 (1) Example 19 Peak 2 >1 (1) Example 19 Peak 3 0.002 (1)Example 19 Peak 4 0.054 (1) Example 20 Peak 1 0.294 (1) Example 20 Peak2 0.038 (2) Example 21 Peak 1 0.142 (2) Example 21 Peak 2 0.008 (2)Example 22 0.222 (2) Example 23 ( 1.26 (1)

Interferon-β Induction: THP-1 ISG Reporter Cell Line

THP-1 Lucia™ ISG cells (InvivoGen) express the secreted luciferase“Lucia” reporter gene under the control of an IRF-inducible compositepromotor comprised of five interferon response elements. THP-1 Lucia™ISG cells were grown in RPMI media plus 2 mM L-glutamine, 10% fetalbovine serum, and 0.5% Pen-Strep. Hygromycin B and Zeocin were presentto maintain stable transfection. 10⁴ cells were seeded in 96-well platesand incubated overnight 37° C., 5% CO₂. 50 μL of serial dilutedcompounds in media (final 0.5% DMSO) was and incubated for an additional24 hours. After incubation, the plates were centrifuged at 2000 rpm for10 min. 50 μl of cell culture supernatant of each well was transferredto a white, opaque 96-well plate. One pouch of QUANTI-Luc™ (InvivoGen)powder was prepared in 25 mL of endotoxin-free water and 100 μL ofprepared warm QUANTI-Luc solution were added to each well containing thesupernatant. The luminescence signal was measured using a Perkin-ElmerEnvision microplate reader. Data were normalized to “% effect” with apositive control STING agonist that was known to maximize the luciferasesignal and the negative control DMSO. Interferon-3 induction data isprovided in Table 3.

TABLE 3 IFN-β induction EC50 IFN-β THP-1 Mean μM reporter (N) Example 1Peak 3 10.12 (6) Example 1 Peak 4 8.49 (6) Example 2 Peak 1 ( 3..08 (4)Example 2 Peak 2 7.9 (4) Example 2 Peak 3 0.93 (9) Example 2 Peak 4 3.02(7) Example 3 Peak 1 6.52 (3) Example 3 Peak 2 22.9 (3) Example 3 Peak 32.1 (2) Example 3 Peak 4 4.5 (3) Example 7 Peak 1 13.8 (1) Example 7Peak 2 >30 (1) Example 7 Peak 3 4.6 (2) Example 7 Peak 4 7.4 (2) Example13 Peak 1 >100 (2) Example 13 Peak 2 >100 (2) Example 14 31.17 (4)Example 15 Peak 1 9.89 (4) Example 15 Peak 2 5.19 (4) Example 16 Peak 131.41 (3) Example 16 Peak 2 14.88 (4) Example 17 Peak 1 10.43 (2)Example 17 Peak 2 3.43 (4) Example 18 0.98 (1) Example 19 Peak 1 5.33(2) Example 19 Peak 2 >100 (1) Example 19 Peak 3 4.39 (1) Example 19Peak 4 8.82 (2) Example 20 Peak 1 80.84 (2) Example 20 Peak 2 7.54 (4)Example 21 Peak 1 >30 (1) Example 21 Peak 2 28.5 (1) Example 22 >30 (1)Example 23 >100 (1)THP-1 Cell Reporter Assay with Different Human STING Polymorphisms toMeasure Type I Interferon Activity

The wild-type (WT) STING allele has been reported to have an additional4 different single nucleotide polymorphisms (SNPs) in the humanpopulation that can affect its response. These SNPs are known asR71H-G230A-R293Q (HAQ), R232H, G230A-R293Q (AQ), and R293Q. In order totest whether indicated compounds can activate all five human STINGalleles representing >98% of the human population, THP-1-Dual KO-STINGcells (InvivoGen) were individually transduced with a lentiviruscontaining one of the human STING alleles (Genecopoeia). Transducedcells were selected and expression of STING was confirmed by westernblot (data not shown). Selected cells were cultured and harvested in 50mL conical tubes, counted using a BC Vi-flow and diluted toconcentration of 7.4×10⁵ cell/ml. 135 μl of diluted cells weretransferred to a 96 well plate (100,000 cells/well) and incubated at 37°C. in a CO₂ incubator for 3 to 4 hours. Next, 15 uL of serially dilutedtest compound were added to each well for stimulation, the platecontaining cells and compounds was further incubated at 37° C. and 5%CO₂ for 24 hours. After incubation, the plates were centrifuged at 2000rpm for 10 min. 50 μl of cell culture supernatant of each well wastransfered to a white, opaque 96 well plate. QUANTI-Luc™ (InvivoGen)powder was prepared in 25 mL of endotoxin-free water and 100 uL ofprepared warm QUANTI-Luc solution were added to each well containingculture supernatant and the luminescence signal was measured immediatelyusing a Perkin Elmer Enspire microplate reader (0.2 sec). RLU wasobtained by raw value. THP-1 cell reporter assay date is provided inTable 4.

TABLE 4 THP-1 ISG THP-1 ISG THP-1 ISG THP-1 ISG THP-1 ISG WT R232H HAQAQ R293Q Compound EC50 (μM) EC50 (μM) EC50 (μM) EC50 (μM) EC50 (μM)Example 1 Peak 3 >50 (N = 2) >50 (N = 3) 32.56 (N = 2) 15.38 (N = 2) >50(N = 2) Example 1 Peak 4 >50 (N = 2) >50 (N = 3) 25.47 (N = 2) 23.93 (N= 2) >50 (N = 2) Example 2 Peak 2 >50 (N = 2) >50 (N = 2) >50 (N= 1) >50 (N = 1) >50 (N = 1) Example 2 Peak 3 7.58 (N = 3) 12.67 (N = 3)16.56 (N = 3) 6.52 (N = 3) 7.16 (N = 3) Example 2 Peak 4 5.79 (N = 4)12.92 (N = 4) 12.58 (N = 4) 5.33 (N = 4) 7.57 (N = 4) Example 2 Peak 15.83 (N = 2) 3.26 (N = 2) >50 (N = 1) 7.32 (N = 2) 6.63 (N = 2) Example14 27.62 (N = 3) >50 (N = 3) 27.13 (N = 3) 26.10 (N = 3) >50 (N = 2)Example 16 Peak 1 23.63 (N = 3) >50 (N = 3) 29.32 (N = 3) 30.69 (N =3) >50 (N = 3) Example 16 Peak 2 16.37 (N = 3) >50 (N = 3) 16.84 (N = 3)12.96 (N = 3) 27.14 (N = 3) Example 15 Peak 2 6.48 (N = 3) >50 (N = 3)9.75 (N = 3) 6.51 (N = 3) 14.40 (N = 3) Example 15 Peak 1 25.77 (N =2) >50 (N = 3) 18.37 (N = 3) 12.58 (N = 3) 24.66 (N = 3) Example 17 Peak1 9.97 (N = 3) >50 (N = 3) 12.80 (N = 3) 6.80 (N = 3) 19.39 (N = 3)Example 17 Peak 2 2.22 (N = 3) 7.34 (N = 3) 4.86 (N = 3) 2.67 (N = 3)4.17 (N = 3)

Phosphorylation of IRF3: THP-1 or OVCAR4 Cell ELISA

STING activation results in recruitment of TBK1 and phosphorylation ofIRF3 transcription factor before induction of type I interferons. THP-1cells (InvivoGen) or OVCAR4 cells (Pfizer Cell Bank) were grown in RPMImedia plus 2 mM L-glutamine, 10% fetal bovine serum, and 0.5% Pen-Strep.10⁴ cells were seeded in 96-well plates and incubated overnight 37° C.,5% CO2. Compounds serial diluted compounds in media (final 0.5% DMSO)were added to the cells and incubated for an additional 3 hours. Afterincubation, the plates were centrifuged at 2000 rpm for 5 min. The cellswere then lysed in 100 μl RIPA buffer and vortexed for 30 minutes atroom temperature. 25 μl of lysate was then transferred to clearpolystyrene High Bind plates that had been previously coated with mouseanti-human IRF-3 capture antibody (BD Pharmigen), and allowed toincubate at 4° C. for 16 hours. The plates were then washed andincubated with rabbit anti-phospho-IRF3 detection antibody (CellSignaling Technologies) for 1.5 hours at room temperature. Finally, anHRP-linked secondary antibody (Cell Signaling Technologies) was addedfor 30 min before the Glo Substrate Reagent (R&D Systems) was usedgenerate the luminescent signal. The signal was measured using aPerkin-Elmer Envision microplate reader. Data were normalized to “%effect” with a positive control STING agonist that was known to maximizethe phosphorylated IRF3 signal and the negative control was DMSO. IRF3Phosphorylation data is provided in Tables 5 and 6.

TABLE 5 pIRF EC50 pIRF3 ELISA Mean μM THP-1 (N) Example 1 Peak 3 25.6(5) Example 1 Peak 4 39.2 (5) Example 2 Peak 1 58.4 (3) Example 2 Peak2 >100 (3) Example 2 Peak 3 13.66 (10) Example 2 Peak 4 23.68 (5)Example 3 Peak 1 >100 (2) Example 3 Peak 2 >100 (2) Example 3 Peak 320.5 (2) Example 3 Peak 4 30.69 (3) Example 7 Peak 1 ND Example 7 Peak 2ND Example 7 Peak 3 ND Example 7 Peak 4 ND Example 13 Peak 1 >100 (2)Example 13 Peak 2 >100 (2) Example 14 >100 (3) Example 15 Peak 1 >100(3) Example 15 Peak 2 31.6 (3) Example 16 Peak 1 >100 (3) Example 16Peak 2 >100 (3) Example 17 Peak 1 92.8 (1) Example 17 Peak 2 18.54 (3)Example 18 9.04 (1) Example 19 Peak 1 56.8 (1) Example 19 Peak 2 >100(1) Example 19 Peak 3 30.9 (1) Example 19 Peak 4 66.0 (1) Example 20Peak 1 >100 (1) Example 20 Peak 2 62.55 (3) Example 21 Peak 1 ND Example21 Peak 2 ND Example 22 ND Example 23 >100 (2)

TABLE 6 pIRF EC50 pIRF3 ELISA Mean μM OVCAR4 (N) Example 14 39.27 (3)Example 15 Peak 1  51.5 (2) Example 15 Peak 2  2.76 (3) Example 16 Peak1  57.0 (3) Example 16 Peak 2 71.46 (3) Example 17 Peak 1 46.37 (2)Example 17 Peak 2  8.06 (3)

We claim:
 1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein each J isindependently oxygen (O) or sulfur (S); R¹ is selected from:

R² is selected from:

W is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion; Xis OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;each Y is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Y substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms; each Z is independently selected from hydrogen, halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Zsubstituents join to form a 3-5 membered spirocyclic ring systemcomprising 0-1 heteroatoms; and R³ and R⁴ are each independentlyhydrogen or C₁-C₆ alkyl.
 2. A compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein R¹ is selectedfrom:

R² is selected from:

W is OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion; Xis OH, SH, O⁻M⁺ or S⁻M⁺, where M⁺ represents a cationic counter-ion;each Y is independently selected from hydrogen, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Y substituents jointo form a 3-5 membered spirocyclic ring system comprising 0-1heteroatoms; each Z is independently selected from hydrogen, halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, N(R³)₂, and OR⁴, or the two Zsubstituents join to form a 3-5 membered spirocyclic ring systemcomprising 0-1 heteroatoms; and R³ and R⁴ are each independentlyhydrogen or C₁-C₆ alkyl.
 3. The compound or salt of claim 1, wherein M⁺is selected from the group consisting of sodium, potassium, calcium,ammonium, triethylammonium, trimethylammonium and magnesium.
 4. Thecompound or salt of claim 1, wherein each counter-ion M⁺ is the same. 5.The compound or salt of claim 1, wherein R¹ is

and R² is


6. The compound or salt of claim 1, wherein R¹ is

and R² is


7. The compound or salt of claim 1, wherein R¹ is

and R² is


8. The compound or salt of claim 1, wherein R¹ is

and R² is


9. The compound or salt of claim 1, wherein R¹ is

and R² is


10. The compound or salt of claim 1, wherein R¹ is

and R² is


11. The compound or salt of claim 1, wherein R¹ is

and R² is


12. The compound or salt of claim 1, wherein R¹ is

and R² is


13. The compound or salt of claim 1, wherein R¹ is

and R² is


14. The compound or salt of claim 1, wherein R¹ is

and R² is


15. The compound or salt of claim 1, wherein R¹ is

and R² is


16. The compound or salt of claim 1, wherein R¹ is

and R² is


17. The compound or salt of claim 1, wherein R¹ is

and R² is


18. The compound or salt of claim 1, wherein one or both Y is halogen.19. The compound or salt of claim 1, wherein one Y is hydrogen and theother Y is a halogen.
 20. The compound or salt of claim 19, wherein saidhalogen is fluorine.
 21. The compound or salt of claim 1, wherein one Zis hydrogen and the other Z is OR⁴.
 22. The compound or salt of claim 1,wherein W is —SH and X is —SH.
 23. The compound or salt of claim 1,wherein W is —OH and X is —OH.
 24. The compound or salt of claim 1,wherein W is —SH and X is —OH.
 25. The compound or salt of claim 1,wherein W is —OH and X is —SH.
 26. A compound selected from:

or a pharmaceutically acceptable salt thereof.
 27. A compound selectedfrom:

or a pharmaceutically acceptable salt thereof.
 28. A compound selectedfrom:

or a pharmaceutically acceptable salt thereof.
 29. A single diastereomerof a compound or salt according to any one of claims 1, 2 and 26-28. 30.A pharmaceutical composition comprising a compound or salt according toany one of claims 1 and 26, and a pharmaceutically acceptable carrier.31. A pharmaceutical composition comprising a compound or salt accordingto any one of claims 1 and 26, wherein said compound is a component ofan antibody-drug conjugate.
 32. A pharmaceutical composition comprisinga compound or salt according to any one of claims 1 and 26, wherein saidcompound is a component of a particle-based delivery system.
 33. Amethod of treating abnormal cell growth in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound or salt according to any one of claims 1 and 26.34. The method of claim 33, wherein said compound or salt is a componentof an antibody-drug conjugate.
 35. The method of claim 33, wherein saidcompound or salt is a component of a particle-based delivery system. 36.The method of claim 33, wherein the abnormal cell growth is cancer. 37.The method of claim 36, wherein the cancer is lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, colon cancer, breast cancer,uterine cancer, carcinoma of the fallopian tubes, carcinoma of theendometrium, carcinoma of the cervix, carcinoma of the vagina, carcinomaof the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, chronic or acute leukemia, lymphocytic lymphomas,cancer of the bladder, cancer of the kidney or ureter, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, or pituitary adenoma.
 38. Use of a compound or saltaccording to any one of 1 and 26 for the preparation of a medicamentuseful in the treatment of abnormal cell growth in a mammal.
 39. The useaccording to claim 38, wherein said abnormal cell growth is cancer. 40.The use according to claim 39, wherein the cancer is lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, chronic or acuteleukemia, lymphocytic lymphomas, cancer of the bladder, cancer of thekidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis,neoplasms of the central nervous system (CNS), primary CNS lymphoma,spinal axis tumors, brain stem glioma, or pituitary adenoma.
 41. Amethod of upregulating the activity of STING in a mammal, comprising thestep of administering to said mammal an effective amount of a compoundor salt according to any one of 1 and
 26. 42. A method of increasinginterferon-beta levels in a mammal, comprising the step of administeringto said mammal an effective amount of a compound or salt according toany one of 1 and 26.